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Coronavirus (CoV) possesses numerous functional cis-acting elements in its positive-strand genomic RNA. Although most of these RNA structures participate in viral replication, the functions of RNA structures in the genomic RNA of CoV in viral replication remain unclear. In this study, we investigated the functions of the higher-order RNA stem-loop (SL) structures SL5B, SL5C, and SL5D in the ORF1a coding region of Middle East respiratory syndrome coronavirus (MERS-CoV) in viral replication. Our approach, using reverse genetics of a bacterial artificial chromosome system, revealed that SL5B and SL5C play essential roles in the discontinuous transcription of MERS-CoV. In silico analyses predicted that SL5C interacts with a bulged stem-loop (BSL) in the 3′ untranslated region, suggesting that the RNA structure of SL5C is important for viral RNA transcription. Conversely, SL5D did not affect transcription, but mediated the synthesis of positive-strand genomic RNA. Additionally, the RNA secondary structure of SL5 in the revertant virus of the SL5D mutant was similar to that of the wild-type, indicating that the RNA structure of SL5D can finely tune RNA replication in MERS-CoV. Our data indicate novel regulatory mechanisms of viral RNA transcription and replication by higher-order RNA structures in the MERS-CoV genomic RNA. [ABSTRACT FROM AUTHOR]

Sleep disturbances are common features of neurodegenerative disorders including Huntington's disease (HD). Sleep and circadian disruptions are recapitulated in animal models, providing the opportunity to evaluate the effectiveness of circadian interventions as countermeasures for neurodegenerative disease. For instance, time restricted feeding (TRF) successfully improved activity rhythms, sleep behavior and motor performance in mouse models of HD. Seeking to determine if these benefits extend to physiological measures of sleep, electroencephalography (EEG) was used to measure sleep/wake states and polysomnographic patterns in male and female wild-type (WT) and bacterial artificial chromosome transgenic (BACHD) adult mice, under TRF and ad lib feeding (ALF). Our findings show that male, but not female, BACHD mice exhibited significant changes in the temporal patterning of wake and non-rapid eye movement (NREM) sleep. The TRF intervention reduced the inappropriate early morning activity by increasing NREM sleep in the male BACHD mice. In addition, the scheduled feeding reduced sleep fragmentation (# bouts) in the male BACHD mice. The phase of the rhythm in rapid-eye movement (REM) sleep was significantly altered by the scheduled feeding in a sex-dependent manner. The treatment did impact the power spectral curves during the day in male but not female mice regardless of the genotype. Sleep homeostasis, as measured by the response to six hours of gentle handling, was not altered by the diet. Thus, TRF improves the temporal patterning and fragmentation of NREM sleep without impacting sleep homeostasis. This work adds critical support to the view that sleep is a modifiable risk factor in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications. [ABSTRACT FROM AUTHOR]

The aberrant expression of specific long noncoding RNAs (lncRNAs) has been associated with cognitive and psychiatric disorders. Although a growing number of lncRNAs are now known to regulate neural cell development and function, relatively few lncRNAs have been shown to underlie animal behavior. Pnky is an evolutionarily conserved, neural lncRNA that regulates brain development. Using mouse genetic strategies, we show that Pnky has sex-specific roles in mouse behavior and that this lncRNA can underlie specific behavior by functioning in trans. Male Pnky-knockout mice have decreased context generalization in a paradigm of associative fear learning and memory. In female Pnky-knockout mice, there is an increase in the acoustic startle response, a behavior that is altered in affective disorders. Remarkably, expression of Pnky from a bacterial artificial chromosome transgene decreases the acoustic startle response in female Pnky-knockout mice, demonstrating that Pnky can modulate specific animal behavior by functioning in trans. More broadly, these studies illustrate how specific lncRNAs can underlie cognitive and mood disorders. Relatively few lncRNAs have been shown to regulate animal behavior. Here, Saha et al. use mouse genetic methods to show that the lncRNA Pnky can underlie specific mouse behavior by functioning in trans. [ABSTRACT FROM AUTHOR]

Conventional gene cloning methods always require tedious processes confined by PCR conditions and restriction sites, which might result in a large number of redundant fragments in the constructs. These shortages can be overcome by using DNA recombination methods. Homologous recombination in Escherichia coli cells was used to develop positive/negative cassette for phage λ red operon recombination. In the bi-functional cassette, the Ala294Gly α-subunit mutant of phenylalanyl-tRNA synthetase (mPheS) was fused with the kanamycin-resistant (KanR) cassette under the control of the promoter of the E. coli gene NEOKAN as a single open reading frame (ORF) by PCR. KanR was utilized as a positive selection marker, while the mutant gene mPheS served as a negative selection marker. Two-step red-mediated recombination was used to replace the third exon of TMEM18 in the BAC clone RP23-25C13 with eGFP. Initially, the third TMEM18 exon in the BAC was replaced with the NEOKAN-mPheS-KanR cassette, and 40% of the clones on the positive selection plate (LB plate with kanamycin) were positive. The second step involved replacing the NEOKAN-mPheS-KanR cassette with eGFP in the modified BAC, and 60% of the clones on the negative plate (p-Cl-phe plate) were positive. Following the two steps, eGFP was used to replace the third exon of TMEM18 in the BAC. The NEOKAN-mPheS-KanR could be a promising choice as a positive/negative selection cassette for red recombination to establish constructs and make gene manipulation on BAC more applicable and effortless. [ABSTRACT FROM AUTHOR]

The genome organization of woodpeckers has several distinctive features e.g., an uncommon accumulation of repetitive sequences, enlarged Z chromosomes, and atypical diploid numbers. Despite the large diversity of species, there is a paucity of detailed cytogenomic studies for this group and we thus aimed to rectify this. Genome organization patterns and hence evolutionary change in the microchromosome formation of four species (Colaptes campestris, Veniliornis spilogaster, Melanerpes candidus, and Picumnus nebulosus) was established through fluorescence in situ hybridization using bacterial artificial chromosomes originally derived from Gallus gallus and Taeniopygia guttata. Findings suggest that P. nebulosus (2n = 110), which was described for the first time, had the most basal karyotype among species of Picidae studied here, and probably arose as a result of fissions of avian ancestral macrochromosomes. We defined a new chromosomal number for V. spilogaster (2n = 88) and demonstrated microchromosomal rearrangements involving C. campestris plus a single, unique hitherto undescribed rearrangement in V. spilogaster. This comprised an inversion after a fusion involving the ancestral microchromosome 12 (homologous to chicken microchromosome 12). We also determined that the low diploid number of M. candidus is related to microchromosome fusions. Woodpeckers thus exhibit significantly rearranged karyotypes compared to the putative ancestral karyotype. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV), a flavivirus transmitted by mosquitoes, has caused epidemics and severe neurological diseases in Asian countries. In this study, we developed a cDNA infectious clone, pBAC JYJEV3, of the JEV genotype 3 strain (EF571853.1) using a bacterial artificial chromosome (BAC) vector. The constructed infectious clone was transfected into Vero cells, where it exhibited infectivity and induced cytopathic effects akin to those of the parent virus. Confocal microscopy confirmed the expression of the JEV envelope protein. Comparative analysis of growth kinetics revealed similar replication dynamics between the parental and recombinant viruses, with peak titers observed 72 h post-infection (hpi). Furthermore, plaque assays demonstrated comparable plaque sizes and morphologies between the viruses. Cryo-electron microscopy confirmed the production of recombinant virus particles with a morphology identical to that of the parent virus. Immunization studies in mice using inactivated parental and recombinant viruses revealed robust IgG responses, with neutralizing antibody production increasing over time. These results showcase the successful generation and characterization of a recombinant JEV3 virus and provide a platform for further investigations into JEV pathogenesis and vaccine development. [ABSTRACT FROM AUTHOR]

Examinations of ovarian cancer cells require the ability to identify tumor cells. Array-based comparative genome hybridization (aCGH) on 30 ovarian carcinomas (OC) identified three genomic loci (8q24.23; 17p12; 18q22.3) over- or under-represented in OC. A fluorescence in situ hybridization (FISH) probe of these three loci is intended to identify tumor cells by their signal pattern deviating from a diploid pattern. Human DNA from these three loci is isolated from bacterial artificial chromosomes (BAC), amplified and labeled with fluorescent dyes. After a standard FISH procedure, 71 OC suspensions from primary tumors, three OC cell lines, three lymphocyte suspensions, and one mesenchymal cell line LP-3 are analyzed with a fluorescence microscope. On average, 15% of the lymphocytes deviate from the expected diploid signal pattern, giving a cut-off of 36%. If this value is exceeded, tumor cells are detected. The mesenchymal cell line LP-3 shows only 21% as a negative control. The OC cell lines as positive controls exceed this value at 38%, 67%, and 54%. Of the 71 OC primary cultures, four cases fell below this cut-off as false negatives. In the two-sample t-test, the percentages of conspicuous signal patterns differ significantly. [ABSTRACT FROM AUTHOR]

The Cuculiformes are a family of over 150 species that live in a range of habitats, such as forests, savannas, and deserts. Here, bacterial artificial chromosome (BAC) probes (75 from chicken and 14 from zebra finch macrochromosomes 1–10 +ZW and for microchromosomes 11–28 (except 16)) were used to investigate chromosome homologies between chicken and the squirrel cuckoo (Piaya cayana). In addition, repetitive DNA probes were applied to characterize the chromosome organization and to explore the role of these sequences in the karyotype evolution of P. cayana. We also applied BAC probes for chicken chromosome 17 and Z to the guira cuckoo (Guira guira) to test whether this species has an unusual Robertsonian translocation between a microchromosome and the Z chromosome, recently described in the smooth-billed ani (Crotophaga ani). Our results revealed extensive chromosome reorganization with inter- and intrachromosomal rearrangements in P. cayana, including a conspicuous chromosome size and heterochromatin polymorphism on chromosome pair 20. Furthermore, we confirmed that the Z-autosome Robertsonian translocation found in C. ani is also found in G. guira, not P. cayana. These findings suggest that this translocation occurred prior to the divergence between C. ani and G. guira, but after the divergence with P. cayana. [ABSTRACT FROM AUTHOR]

In 2021, a highly virulent strain of duck enteritis virus (DEV), designated as DEV XJ, was isolated from Zhejiang, China, and its complete genome, spanning 162,234 bp with 78 predicted open reading frames (ORFs), was sequenced. While showing relative homology to the DEV CV strain, DEV XJ exhibited distinctions in 38 ORFs, including various immunogenic and virulence-related genes. Amino acid variation analysis, focusing on UL6 and LORF3, indicated a high degree of homology between DEV XJ and the 2085 strain from Europe, as well as the DEV DP-AS-Km-19 strain from India. Subsequently, a full-length infectious bacterial artificial chromosome clone (BAC) of DEV XJ was successfully constructed to delve into the pathogenic mechanisms of this virulent strain. XJ BAC demonstrated substantial similarity to the parental DEV XJ in both in vitro growth properties and the induction of typical pathogenic symptoms in sheldrakes. Furthermore, the US3, LORF3, UL21, and UL36 genes were individually deleted using a two-step RED recombination approach based on the infectious BAC clone. Our findings revealed that the UL21 and UL36 genes play crucial roles in viral proliferation. Although the US3 and LORF3 genes were dispensable for viral replication and cell-to-cell transmission in vitro, they attenuated the replication and transmission efficiency of DEV compared to the WT. In summary, this study accomplished the whole-genome sequencing of a clinically virulent DEV strain and the successful construction of an infectious DEV XJ clone. Moreover, the functional roles of the above-mentioned mutant genes were preliminarily explored through the analysis of their in vitro biological characteristics. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces direct cytopathic effects, complicating the establishment of low-cytotoxicity cell culture models for studying its replication. We initially developed a DNA vector-based replicon system utilizing the CMV promoter to generate a recombinant viral genome bearing reporter genes. However, this system frequently resulted in drug resistance and cytotoxicity, impeding model establishment. Herein, we present a novel cell culture model with SARS-CoV-2 replication induced by Cre/LoxP-mediated DNA recombination. An engineered SARS-CoV-2 transcription unit was subcloned into a bacterial artificial chromosome (BAC) vector. To enhance biosafety, the viral spike protein gene was deleted, and the nucleocapsid gene was replaced with a reporter gene. An exogenous sequence was inserted within NSP1 as a modulatory cassette that is removable after Cre/LoxP-mediated DNA recombination and subsequent RNA splicing. Using the PiggyBac transposon strategy, the transcription unit was integrated into host cell chromatin, yielding a stable cell line capable of inducing recombinant SARS-CoV-2 RNA replication. The model exhibited sensitivity to the potential antivirals forsythoside A and verteporfin. An innovative inducible SARS-CoV-2 replicon cell model was introduced to further explore the replication and pathogenesis of the virus and facilitate screening and assessment of anti-SARS-CoV-2 therapeutics. [ABSTRACT FROM AUTHOR]

Tissue-specific gene manipulations are widely used in genetically engineered mouse models. A single recombinase system, such as the one using Alb-Cre, has been commonly used for liver-specific genetic manipulations. However, most diseases are complex, involving multiple genetic changes and various cell types. A dual recombinase system is required for conditionally modifying different genes sequentially in the same cell or inducing genetic changes in different cell types within the same organism. A FlpO cDNA was inserted between the last exon and 3′-UTR of the mouse albumin gene in a bacterial artificial chromosome (BAC-Alb-FlpO). The founders were crossed with various reporter mice to examine the efficiency of recombination. Liver cancer tumorigenesis was investigated by crossing the FlpO mice with FSF-KrasG12D mice and p53frt mice (KPF mice). BAC-Alb-FlpO mice exhibited highly efficient recombination capability in both hepatocytes and intrahepatic cholangiocytes. No recombination was observed in the duodenum and pancreatic cells. BAC-Alb-FlpO-mediated liver-specific expression of mutant KrasG12D and conditional deletion of p53 gene caused the development of liver cancer. Remarkably, liver cancer in these KPF mice manifested a distinctive mixed hepatocellular carcinoma and cholangiocarcinoma phenotype. A highly efficient and liver-specific BAC-Alb-FlpO mouse model was developed. In combination with other Cre lines, different genes can be manipulated sequentially in the same cell, or distinct genetic changes can be induced in different cell types of the same organism. NEW & NOTEWORTHY: A liver-specific Alb-FlpO mouse line was generated. By coupling it with other existing CreERT or Cre lines, the dual recombinase approach can enable sequential gene modifications within the same cell or across various cell types in an organism for liver research through temporal and spatial gene manipulations. [ABSTRACT FROM AUTHOR]

We have previously developed a bacterial artificial chromosome (BAC)‐vectored SARS‐CoV‐2 replicon, namely BAC‐CoV2‐Rep, which, upon transfection into host cells, serves as a transcription template for SARS‐CoV‐2 replicon mRNA to initiate replicon replication and produce nanoluciferase (Nluc) reporter from the subgenomic viral mRNA. However, an inherent issue of such DNA‐launched replicon system is that the nascent full‐length replicon transcript undergoes process by host RNA splicing machinery, which reduces replicon replication and generates spliced mRNA species expressing NLuc reporter independent of replicon replication. To mitigate this problem, we employed Isoginkgetin, a universal eukaryotic host splicing inhibitor, to treat cells transfected with BAC‐CoV2‐Rep. Isoginkgetin effectively increased the level of full‐length replicon transcripts while concurrently reducing the level of Nluc signal derived from spliced replicon mRNA, making the Nluc reporter signal more correlated with replicon replication, as evidenced by treatment with known SARS‐CoV‐2 replication inhibitors including Remdesivir, GC376, and EIDD‐1931. Thus, our study emphasizes that host RNA splicing is a confounding factor for DNA‐launched SARS‐CoV‐2 replicon systems, which can be mitigated by Isoginkgetin treatment. [ABSTRACT FROM AUTHOR]

The Neotropical deer genus Mazama is characterized by homoplastic morphological characters, a high karyotypic diversity, and a polyphyletic condition. The species of the genus have been recovered into two multigeneric lineages, the subtribes Odocoileina and Blastocerina, of the tribe Odocoileini (New World deer) in the family Cervidae. Within the Blastocerina, gray brockets include two non-sister species, Subulo gouazoubira, occurring south of the Amazon region, and Passalites nemorivagus, occurring in the Guianas and in the Amazon region. We clarify the taxonomic status and phylogenetic position of Mazama americana citus Osgood, 1912 (referred to as either S. gouazoubira or P. nemorivagus by other authors). We collected a topotype of M. a. citus from the eastern shore of Lake Maracaibo, Venezuela, characterize it morphologically and cytogenetically (conventional banding and fluorescence in situ hybridization), and carry out a phylogenetic analysis of its whole mitogenome and Cytb alongside two additional specimens of M. a. citus from northwestern Venezuela. Our analyses reveal the topotype to be a large gray brocket with a cinnamon band above the eyes and 2n = 61 and FN = 70 karyotype. Using cattle whole chromosome painting and bacterial artificial chromosome X probes, we determined its karyotype to differ in at least 10 rearrangements from that of S. gouazoubira. Bayesian inference recovers M. a. citus within the Blastocerina subtribe, separated phylogenetically from other gray brockets (100% branch value), revealing the Osgood's gray brocket to be a valid species that should be assigned to a new genus. We propose the generic name Bisbalus, with Bisbalus citus (Osgood, 1912) as the type species. [ABSTRACT FROM AUTHOR]

The SpTransformer (SpTrf) gene family in the purple sea urchin, Strongylocentrotus purpuratus, encodes immune response proteins. The genes are clustered, surrounded by short tandem repeats, and some are present in genomic segmental duplications. The genes share regions of sequence and include repeats in the coding exon. This complex structure is consistent with putative local genomic instability. Instability of the SpTrf gene cluster was tested by 10 days of growth of Escherichia coli harboring bacterial artificial chromosome (BAC) clones of sea urchin genomic DNA with inserts containing SpTrf genes. After the growth period, the BAC DNA inserts were analyzed for size and SpTrf gene content. Clones with multiple SpTrf genes showed a variety of deletions, including loss of one, most, or all genes from the cluster. Alternatively, a BAC insert with a single SpTrf gene was stable. BAC insert instability is consistent with variations in the gene family composition among sea urchins, the types of SpTrf genes in the family, and a reduction in the gene copy number in single coelomocytes. Based on the sequence variability among SpTrf genes within and among sea urchins, local genomic instability of the family may be important for driving sequence diversity in this gene family that would be of benefit to sea urchins in their arms race with marine microbes. [ABSTRACT FROM AUTHOR]

Sugarcane (Saccharum spp.) is an important crop for sugar and bioethanol production worldwide. To maintain and increase sugarcane yields in marginal areas, the use of nitrogen (N) fertilizers is essential, but N overuse may result in the leaching of reactive N to the natural environment. Despite the importance of N in sugarcane production, little is known about the molecular mechanisms involved in N homeostasis in this crop, particularly regarding ammonium (NH4+), the sugarcane's preferred source of N. Here, using a sugarcane bacterial artificial chromosome (BAC) library and a series of in silico analyses, we identified an AMMONIUM TRANSPORTER (AMT) from the AMT2 subfamily, sugarcane AMMONIUM TRANSPORTER 3;3 (ScAMT3;3), which is constitutively and highly expressed in young and mature leaves. To characterize its biochemical function, we ectopically expressed ScAMT3;3 in heterologous systems (Saccharomyces cerevisiae and Arabidopsis thaliana). The complementation of triple mep mutant yeast demonstrated that ScAMT3;3 is functional for NH3/H+ cotransport at high availability of NH4+ and under physiological pH conditions. The ectopic expression of ScAMT3;3 in the Arabidopsis quadruple AMT knockout mutant restored the transport capacity of 15N-NH4+ in roots and plant growth under specific N availability conditions, confirming the role of ScAMT3;3 in NH4+ transport in planta. Our results indicate that ScAMT3;3 belongs to the low-affinity transport system (Km 270.9 µM; Vmax 209.3 µmol g-1 root DW h-1). We were able to infer that ScAMT3;3 plays a presumed role in NH4+ source-sink remobilization in the shoots via phloem loading. These findings help to shed light on the functionality of a novel AMT2-type protein and provide bases for future research focusing on the improvement of sugarcane yield and N use efficiency. [ABSTRACT FROM AUTHOR]

Pacific abalone (Haliotis discus hannai) is an economically important marine shellfish for aquaculture and is distributed throughout eastern Asia. Although a lot of genetic breeding work has been carried out, chromosome identification in abalone is still a challenging task. Here, we developed a set of BACs to be chromosome-specific probes in Pacific abalone, and to study chromosome evolution in the related species. Through BAC paired-end sequencing and sequence alignment, we were able to in silico anchor 168 BACs onto 18 pseudochromosomes of Pacific abalone genome. After selecting 42 BACs that contained DNA inserts with minimal repetitive sequences, we validated them through PCR and Fluorescence in situ hybridization (FISH) test. As a result, We obtained specific FISH signals for 26 clones on the chromosomes of Pacific abalone with at least one BAC mapped per chromosome. We also applied the chromosome-specific BAC-FISH probes to a close relative of Pacific abalone, Xishi abalone (H. gigantea), which revealed that chromosome 13 and 15 between the two species underwent a chromosomes rearrangement event. This study provides the first set of chromosome-specific probes for the family Haliotidae, which can serve as an important tool for future cytogenetics and genomics research. [ABSTRACT FROM AUTHOR]

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are fatal neurodegenerative diseases that represent ends of the spectrum of a single disease. The most common genetic cause of FTD and ALS is a hexanucleotide repeat expansion in the C9orf72 gene. Although epidemiological data suggest that traumatic brain injury (TBI) represents a risk factor for FTD and ALS, its role in exacerbating disease onset and course remains unclear. To explore the interplay between traumatic brain injury and genetic risk in the induction of FTD/ALS pathology we combined a mild repetitive traumatic brain injury paradigm with an established bacterial artificial chromosome transgenic C9orf72 (C9BAC) mouse model without an overt motor phenotype or neurodegeneration. We assessed 8–10 week-old littermate C9BACtg/tg (n = 21), C9BACtg/− (n = 20) and non-transgenic (n = 21) mice of both sexes for the presence of behavioural deficits and cerebral histopathology at 12 months after repetitive TBI. Repetitive TBI did not affect body weight gain, general neurological deficit severity, nor survival over the 12-month observation period and there was no difference in rotarod performance, object recognition, social interaction and acoustic characteristics of ultrasonic vocalizations of C9BAC mice subjected to repetitive TBI versus sham injury. However, we found that repetitive TBI increased the time to the return of the righting reflex, reduced grip force, altered sociability behaviours and attenuated ultrasonic call emissions during social interactions in C9BAC mice. Strikingly, we found that repetitive TBI caused widespread microglial activation and reduced neuronal density that was associated with loss of histological markers of axonal and synaptic integrity as well as profound neuronal transactive response DNA binding protein 43 kDa mislocalization in the cerebral cortex of C9BAC mice at 12 months; this was not observed in non-transgenic repetitive TBI and C9BAC sham mice. Our data indicate that repetitive TBI can be an environmental risk factor that is sufficient to trigger FTD/ALS-associated neuropathology and behavioural deficits, but not paralysis, in mice carrying a C9orf72 hexanucleotide repeat expansion. [ABSTRACT FROM AUTHOR]

Objective: Intellectual disability (ID) is a complex, variable, and clinically heterogeneous neurodevelopmental disorder that affects 1% - 3% of the global population. Copy number variations (CNVs) contribute to approximately 15%-20% of ID cases. Array comparative genomic hybridization (aCGH) is the first-line test for diagnosing patients with ID with/without multiple congenital anomalies (MCAs). This study aimed to present CNVs identified in a retrospective aCGH cohort of Turkish patients with ID with/without other medical conditions. Materials and Methods: The study population consisted of 210 patients (139 male, 71 female) aged 2-18 years. aCGH analysis was performed using oligo and bacterial artificial chromosome (BAC)-based microarray platforms. CNVs were interpreted using public databases and literature mining and categorized according to international guidelines. Results: Forty-five CNVs were detected in 38 (18%) patients. Among these CNVs, 21 (46.6%) were pathogenic, 4 (8.8%) were likely pathogenic, and 8 (17.7%) were variants of uncertain clinical significance (VUS). Nineteen CNVs corresponded to rare microdeletion/ microduplication syndromes. Conclusions: This study reports rare CNVs or syndromes among Turkish patients with ID with/without other medical conditions. Data revealed an overall diagnostic rate of 11.43%, which confirms aCGH as the first-line technology allowing geneticists to diagnose complex phenotypes, identify candidate genes involved in ID, and explore novel CNV effects. [ABSTRACT FROM AUTHOR]

Engineering of reverse genetics systems for newly emerged viruses allows viral genome manipulation, being an essential tool for the study of virus life cycle, virus-host interactions and pathogenesis, as well as for the development of effective antiviral strategies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent human coronavirus that has caused the coronavirus disease (COVID-19) pandemic. The engineering of a full-length infectious cDNA clone and a fluorescent replicon of SARS-CoV-2 Wuhan-Hu-1, using a bacterial artificial chromosome, is reported. Viral growth and genetic stability in eleven cell lines were analyzed, showing that both VeroE6 cells overexpressing transmembrane serin protease 2 (TMPRSS2) and human lung derived cells resulted in the optimization of a cell system to preserve SARS-CoV-2 genetic stability. The recombinant SARS-CoV-2 virus and a point mutant expressing the D614G spike protein variant were virulent in a mouse model. The RNA replicon was propagation-defective, allowing its use in BSL-2 conditions to analyze viral RNA synthesis. The SARS-CoV-2 reverse genetics systems developed constitute a useful tool for studying the molecular biology of the virus, the development of genetically defined vaccines and to establish systems for antiviral compounds screening. [ABSTRACT FROM AUTHOR]

Engineering of reverse genetics systems for newly emerged viruses allows viral genome manipulation, being an essential tool for the study of virus life cycle, virus-host interactions and pathogenesis, as well as for the development of effective antiviral strategies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent human coronavirus that has caused the coronavirus disease (COVID-19) pandemic. The engineering of a full-length infectious cDNA clone and a fluorescent replicon of SARS-CoV-2 Wuhan-Hu- 1, using a bacterial artificial chromosome, is reported. Viral growth and genetic stability in eleven cell lines were analyzed, showing that both VeroE6 cells overexpressing transmembrane serin protease 2 (TMPRSS2) and human lung derived cells resulted in the optimization of a cell system to preserve SARS-CoV- 2 genetic stability. The recombinant SARS-CoV-2 virus and a point mutant expressing the D614G spike protein variant were virulent in a mouse model. The RNA replicon was propagation-defective, allowing its use in BSL-2 conditions to analyze viral RNA synthesis. The SARS-CoV-2 reverse genetics systems developed constitute a useful tool for studying the molecular biology of the virus, the development of genetically defined vaccines and to establish systems for antiviral compounds screening. [ABSTRACT FROM AUTHOR]

Since 2011, pseudorabies based on the pseudorabies virus (PRV) variant has emerged as a serious health issue in pig farms in China. The PRV gE/TK or gE/gI/TK deletion strains protect against emerging PRV variants. However, these variants may cause lethal infections in newborn piglets without PRV antibodies. Previous studies have shown that codon deoptimization of a virulence gene causes virus attenuation. Accordingly, we deoptimized US3-S (US3 gene encoding a short isoform that represents approximately 95% of the total US3 transcription) and UL56 genes (first 10 or all codons) of PRV gE/TK deletion strain (PRVΔTK&gE-AH02) to generate six recombinant PRVs through bacterial artificial chromosome technology. In swine testicular cells, recombinant PRVs with all codon deoptimization of US3-S or UL56 genes were grown to lower titers than the parental virus. Notably, US3-S or UL56 with all codon deoptimization reduced mRNA and protein expressions. Subsequently, the safety and immunogenicity of recombinant PRVs with codon deoptimization of US3-S or UL56 are evaluated as vaccine candidates in mice and piglets. The mice inoculated with recombinant PRVs with codon deoptimization of US3-S or UL56 showed exceptional survival ability without severe clinical signs. All codons deoptimized (US3-S and UL56) significantly decreased virus load and attenuated pathological changes in the brains of the mice. Moreover, the protection efficiency offered by recombinant PRVs with codon deoptimization of US3-S or UL56 showed similar effects to PRVΔTK&gE-AH02. Remarkably, the 1-day-old PRV antibody-negative piglets inoculated with PRVΔTK&gE-US3-ST-CD (a recombinant PRV with all codon deoptimization of US3-S) presented no abnormal clinical symptoms, including fever. The piglets inoculated with PRVΔTK&gE-US3-ST-CD showed a high serum neutralization index against the PRV variant. In conclusion, these results suggest using codon deoptimization to generate innovative live attenuated PRV vaccine candidates. [ABSTRACT FROM AUTHOR]

Viral vectors have emerged as powerful tools for delivering and expressing foreign genes, playing a pivotal role in gene therapy. Among these vectors, cytomegalovirus (CMV) stands out as a promising viral vector due to its distinctive attributes including large packaging capacity, ability to achieve superinfection, broad host range, capacity to induce CD8+ T cell responses, lack of integration into the host genome, and other qualities that make it an appealing vector candidate. Engineered attenuated CMV strains such as Towne and AD169 that have a ~15 kb genomic DNA deletion caused by virus passage guarantee human safety. CMV's large genome enables the efficient incorporation of substantial foreign genes as demonstrated by CMV vector-based therapies for SIV, tuberculosis, cancer, malaria, aging, COVID-19, and more. CMV is capable of reinfecting hosts regardless of prior infection or immunity, making it highly suitable for multiple vector administrations. In addition to its broad cellular tropism and sustained high-level gene expression, CMV triggers robust, virus-specific CD8+ T cell responses, offering a significant advantage as a vaccine vector. To date, successful development and testing of murine CMV (MCMV) and rhesus CMV (RhCMV) vectors in animal models have demonstrated the efficacy of CMV-based vectors. These investigations have explored the potential of CMV vectors for vaccines against HIV, cancer, tuberculosis, malaria, and other infectious pathogens, as well as for other gene therapy applications. Moreover, the generation of single-cycle replication CMV vectors, produced by deleting essential genes, ensures robust safety in an immunocompromised population. The results of these studies emphasize CMV's effectiveness as a gene delivery vehicle and shed light on the future applications of a CMV vector. While challenges such as production complexities and storage limitations need to be addressed, ongoing efforts to bridge the gap between animal models and human translation continue to fuel the optimism surrounding CMV-based vectors. This review will outline the properties of CMV vectors and discuss their future applications as well as possible limitations. [ABSTRACT FROM AUTHOR]

The unknown pathogenicity of a significant number of variants found in cancer-related genes is attributed to limited epidemiological data, resulting in their classification as variant of uncertain significance (VUS). To date, Breast Cancer gene-2 (BRCA2) has the highest number of VUSs, which has necessitated the development of several robust functional assays to determine their functional significance. Here we report the use of a humanized-mouse embryonic stem cell (mESC) line expressing a single copy of the human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by BRCA2 exons 3, 18, 19 and all possible single-nucleotide variants in exon 13 and multiplexed these variants for their functional categorization. Specifically, we used a pool of 180-mer single-stranded donor DNA to generate all possible combination of variants. Using a high throughput sequencing-based approach, we show a significant drop in the frequency of non-functional variants, whereas functional variants are enriched in the pool of the cells. We further demonstrate the response of these variants to the DNA-damaging agents, cisplatin and olaparib, allowing us to use cellular survival and drug response as parameters for variant classification. Using this approach, we have categorized 599 BRCA2 variants including 93-single nucleotide variants (SNVs) across the 11 codons, of which 28 are reported in ClinVar. We also functionally categorized 252 SNVs from exon 13 into 188 functional and 60 non-functional variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS. Author summary: The exponential rise in genetic sequencing led to the identification of several missense variants. However, its clinical utility is often limited by our ability to determine the functional impact of genetic variants. This has resulted in the identification of many variant of uncertain significance (VUS), particularly in BRCA2. Several functional assays have been developed to ascertain the impact of VUSs on protein function that can be used to determine their pathogenicity. Our mouse ES cell (mESC)-based method includes generation of individual BRCA2 variants in a bacterial artificial chromosome (BAC) by recombineering and assess their expression in mouse ES cells. These cells can then be used to determine how the variants affect mESC viability and sensitivity to DNA damaging agents. Although the assay has high sensitivity and specificity, the process is time consuming and labor-intensive. To overcome these limitations, here we report the development of a CRISPR-Cas9 based high-throughput approach that can be used to classify multiple BRCA2 variants. Specifically, we utilized the BRCA2-independent single-stranded annealing pathway to efficiently knock-in targeted mutations in mESCs using a library of single-stranded DNA donors. Not only do our results support the ClinVar classification of some variants, but we also assign clinical significance to several VUSs, for which no functional data is currently available. [ABSTRACT FROM AUTHOR]

Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in these drug targets is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein encoded as a small domain at the N terminus of nonstructural protein 3. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and IFN-stimulated gene expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target. [ABSTRACT FROM AUTHOR]

Background: High expression of the Cytokine Receptor-Like Factor 2 (CRLF2) gene has been observed in patients with acute lymphoblastic leukemia BCR-ABL1-like subtype. Currently, there is no commercial system available for the direct detection of the IGH::CRLF2 fusion by fluorescent in situ hybridization (FISH), as there are for many other leukemia-related gene fusions. In an effort to verify the IGH::CRLF2 fusion, some researchers prepare home-grown FISH probes from bacterial artificial chromosome clones flanking the IGH and CRLF2 genes, which is the best alternative to confirm the fusion, however difficult to reproduce in most cytogenetic laboratories. Results: For the direct observation of the IGH::CRLF2 gene fusion we designed a methodological approach requiring the two commercially available IGH and CRLF2 break-apart probes. Conclusions: Our methodological approach allows direct visualization of the IGH::CRLF2 gene fusion and has the potential to be used for identification of other gene fusions. [ABSTRACT FROM AUTHOR]

In this study, we applied bacterial artificial chromosome (BAC) technology with PRVΔTK/gE/gI as the base material to replace the first, central, and terminal segments of the US3 gene with codon-deoptimized fragments via two-step Red-mediated recombination in E. coli GS1783 cells. The three constructed BACs were co-transfected with gI and part of gE fragments carrying homologous sequences (gI+gE'), respectively, in swine testicular cells. These three recombinant viruses with US3 codon de-optimization ((PRVΔTK&gE-US3deop−1, PRVΔTK&gE-US3deop−2, and PRVΔTK&gE-US3deop−3) were obtained and purified. These three recombinant viruses exhibited similar growth kinetics to the parental AH02LA strain, stably retained the deletion of TK and gE gene fragments, and stably inherited the recoded US3. Mice were inoculated intraperitoneally with the three recombinant viruses or control virus PRVΔTK&gEAH02 at a 107.0 TCID50 dose. Mice immunized with PRVΔTK&gE-US3deop−1 did not develop clinical signs and had a decreased virus load and attenuated pathological changes in the lungs and brain compared to the control group. Moreover, immunized mice were challenged with 100 LD50 of the AH02LA strain, and PRVΔTK&gE-US3deop−1 provided similar protection to that of the control virus PRVΔTK&gEAH02. Finally, PRVΔTK&gE-US3deop−1 was injected intramuscularly into 1-day-old PRV-negative piglets at a dose of 106.0 TCID50. Immunized piglets showed only slight temperature reactions and mild clinical signs. However, high levels of seroneutralizing antibody were produced at 14 and 21 days post-immunization. In addition, the immunization of PRVΔTK&gE-US3deop−1 at a dose of 105.0 TCID50 provided complete clinical protection and prevented virus shedding in piglets challenged by 106.5 TCID50 of the PRV AH02LA variant at 1 week post immunization. Together, these findings suggest that PRVΔTK&gE-US3deop−1 displays great potential as a vaccine candidate. [ABSTRACT FROM AUTHOR]

The family Cervidae is the second most diverse in the infraorder Pecora and is characterized by variability in the diploid chromosome numbers among species. X chromosomes in Cervidae evolved through complex chromosomal rearrangements of conserved segments within the chromosome, changes in centromere position, heterochromatic variation, and X-autosomal translocations. The family Cervidae consists of two subfamilies: Cervinae and Capreolinae. Here we build a detailed X chromosome map with 29 cattle bacterial artificial chromosomes of representatives of both subfamilies: reindeer (Rangifer tarandus), gray brocket deer (Mazama gouazoubira), Chinese water deer (Hydropotes inermis) (Capreolinae); black muntjac (Muntiacus crinifrons), tufted deer (Elaphodus cephalophus), sika deer (Cervus nippon) and red deer (Cervus elaphus) (Cervinae). To track chromosomal rearrangements during Cervidae evolution, we summarized new data, and compared them with available X chromosomal maps and chromosome level assemblies of other species. We demonstrate the types of rearrangements that may have underlined the variability of Cervidae X chromosomes. We detected two types of cervine X chromosome—acrocentric and submetacentric. The acrocentric type is found in three independent deer lineages (subfamily Cervinae and in two Capreolinae tribes—Odocoileini and Capreolini). We show that chromosomal rearrangements on the X-chromosome in Cervidae occur at a higher frequency than in the entire Ruminantia lineage: the rate of rearrangements is 2 per 10 million years. [ABSTRACT FROM AUTHOR]

In response to Pi deprivation, phosphate 1 (PHO1) is a significant regulator at trans-eQTL hotspots in Brassica rapa. Brassica rapa short-read sequencing data analysis revealed four PHO1 paralog genes, PHO1_A, PHO1_B, PHO1_C, and PHO1_D, placed in tandem with very high sequence similarity. However, based on short-read genomic sequence data, only three transcripts are accessible. Five bacterial artificial chromosomes (BACs) can be sequenced using a long-read sequencer, which improves de novo assembly and identifies structural variants. The PHO1 gene's quadruplicating tandem positions in the genomic sequence were confirmed by an analysis of long-read data. Transcript analysis identified only three groups of PHO1 paralogs (ortholog AT1G14040 in Arabidopsis), i.e., PHO1_A, PHO1_B, and PHO1_D, expressed in B. rapa leaf tissues under Pi deficiency. PHO1_A, with transcript ID XM_009150437.2, has five different splice variants found. These splice variants' truncated proteins demonstrated PHO1_A's function in P control as opposed to protein encoding. [ABSTRACT FROM AUTHOR]

Macroptilium (Benth.) Urb. is a neotropical legume genus from the subtribe Phaseolinae. The investigated species present a stable chromosome number (2n = 22), but differ in their karyotype formulae, suggesting the presence of chromosome rearrangements. In this work, we comparatively analysed the karyotypes of six species (Macroptilium atropurpureum, Macroptilium bracteatum, Macroptilium erythroloma, Macroptilium gracile, Macroptilium lathyroides, and Macroptilium martii) from the two main clades that form the genus. Heterochromatin distribution was investigated with chromomycin A3 (CMA)/4′,6-diamidino-2-phenylindole (DAPI) staining and fluorescent in situ hybridization was used to localize the 5S and 35S ribosomal DNA (rDNA) sites. Single copy bacterial artificial chromosomes (BACs) previously mapped in the related genera Phaseolus L. and Vigna Savi were used to establish chromosome orthologies and to investigate possible rearrangements among species. CMA+/DAPI− bands were observed, mostly associated with rDNA sites. Additional weak, pericentromeric bands were observed on several chromosomes. Although karyotypes were similar, species could be differentiated mainly by the number and position of the 5S and 35S rDNA sites. BAC markers demonstrated conserved synteny of the main rDNA sites on orthologous chromosomes 6 and 10, as previously observed for Phaseolus and Vigna. The karyotypes of the six species could be differentiated, shedding light on its karyotype evolution. [ABSTRACT FROM AUTHOR]

Background: Cones are essential for color recognition, high resolution, and central vision; therefore cone death causes blindness. Understanding the pathophysiology of each cell type in the retina is key to developing therapies for retinal diseases. However, studying the biology of cone cells in the rod-dominant mammalian retina is particularly challenging. In this study, we used a bacterial artificial chromosome (BAC) recombineering method to knock in the "CreERT2" sequence into the Gnat2 and Arr3 genes, respectively and generated three novel inducible CreERT2 mice with different cone cell specificities. Results: These models (Gnat2CreERT2, Arr3T2ACreERT2, and Arr3P2ACreERT2) express temporally controllable Cre recombinase that achieves conditional alleles in cone photoreceptors. Cre-LoxP recombination can be induced as early as postnatal day (PD) two upon tamoxifen injection at varying efficiencies, ranging from 10 to 15% in Gnat2CreERT2, 40% in Arr3T2ACreERT2, and 100% in Arr3P2ACreERT2. Notably, knocking in the P2A-CreERT2 cassette does not affect cone cell morphology and functionality. Most cone-phototransduction enzymes, including Opsins, CNGA3, etc. are not altered except for a reduction in the Arr3 transcript. Conclusions: The Arr3P2ACreERT2 mouse, an inducible cone-specific Cre driver, is a valuable line in studying cone cell biology, function, as well as its relationship with rod and other retinal cells. Moreover, the Cre activity can be induced by delivering tamoxifen intragastrically as early as PD2, which will be useful for studying retinal development or in rapid degenerative mouse models. [ABSTRACT FROM AUTHOR]

Simple Summary: The Passeriformes order (songbirds) is incredibly diverse in terms of number of species and morphological and ecological diversification, comprising around 60% of all bird species. Despite considerable diversity, the genome organizational structure (i.e., the number and pattern of chromosomes) within Passeriformes is highly conserved, with a chromosome number that remains close to 80 in nearly all species studied. These characteristics raise interesting questions and stimulate curiosity about the genome evolution of this group. Therefore, this study aimed to analyze the organization of the smallest chromosomes (microchromosomes) in four Passeriformes species to understand whether they were rearranged during evolution. This has only recently become possible using fluorescent probes called bacterial artificial chromosomes (BACs) and a technique called fluorescence in situ hybridization (FISH). Our results confirm that the songbirds studied did not rearrange their microchromosomes to any great extent, and this may have contributed to their overall evolutionary success. Passeriformes birds are widely recognized for their remarkable diversity, with over 5700 species described so far. Like most bird species, they possess a karyotype characteristic of modern birds, which includes a bimodal karyotype consisting of a few pairs of macrochromosomes and many pairs of microchromosomes. Although the karyotype is typically 2n = 80, the diploid number can atypically vary greatly, ranging from 56 to approximately 100 chromosomes. In this study, we aimed to understand the extent of conservation of the karyotype's organizational structure within four species of this group using Bacterial Artificial Chromosomes via Fluorescence In Situ Hybridization (BAC-FISH) with microchromosome probes from Chicken (Gallus gallus) or Zebra Finch (Taeniopygia guttata) per microchromosomes (GGA10-28, except GGA16). By examining the chromosome complement of four passerine species—the Streaked Flycatcher (Myiodynastes maculatus), Shiny Cowbird (Molothrus bonariensis), Southern House Wren (Troglodytes aedon), and Double-collared Seedeater (Sporophila caerulescens)—we discovered a new chromosome number for Southern House Wren. Through FISH experiments, we were able to observe the same pattern of microchromosome organization as in the common ancestor of birds. As a result, we propose a new diploid number for Southern House Wren and confirm the conservation status of microchromosome organization, which may confer evolutionary advantages to this group. [ABSTRACT FROM AUTHOR]

Further studies will be necessary to elucidate the effects of TREM2 variants and targeting TREM2 function within the context of A -induced tau seeding and spreading as well as in the phase of tauopathy that is closely linked with neurodegeneration. What is it about the H157Y mutation and relationship between microglia, TREM2, sTREM2 and A that results in decreased A when assessed at a late stage of disease? Most notably, rare variants, such as the R47H, R62H and H157Y mutations, in the Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) gene increase risk for late-onset AD [[1]]. Keywords: Alzheimer's disease; TREM2; Amyloid-beta; Microglia EN Alzheimer's disease TREM2 Amyloid-beta Microglia 1 4 4 04/06/23 20230405 NES 230405 Recent genetic studies have highlighted the central role of the innate immune system in Alzheimer's disease (AD) by identifying several risk variants in genes that are exclusively expressed within microglia in the brain. [Extracted from the article]

Human immunodeficiency virus (HIV) induced AIDS causes a large number of infections and deaths worldwide every year, still no vaccines are available to prevent infection. Recombinant herpes simplex virus type 1 (HSV-1) vector-based vaccines coding the target proteins of other pathogens have been widely used for disease control. Here, a recombinant virus with HIV-1 gp160 gene integration into the internal reverse (IR) region-deleted HSV-1 vector (HSV-BAC), was obtained by bacterial artificial chromosome (BAC) technology, and its immunogenicity investigated in BALB/c mice. The result showed similar replication ability of the HSV-BAC-based recombinant virus and wild type. Furthermore, humoral and cellular immune response showed superiority of intraperitoneal (IP) administration, compared to intranasally (IN), subcutaneous (SC) and intramuscularly (IM), that evidenced by production of significant antibody and T cell responses. More importantly, in a prime-boost combination study murine model, the recombinant viruses prime followed by HIV-1 VLP boost induced stronger and broader immune responses than single virus or protein vaccination in a similar vaccination regimen. Antibody production was sufficient with huge potential for viral clearance, along with efficient T-cell activation, which were evaluated by the enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FC). Overall, these findings expose the value of combining different vaccine vectors and modalities to improve immunogenicity and breadth against different HIV-1 antigens. [ABSTRACT FROM AUTHOR]

Background: Reverse genetics systems to rescue viruses from modified DNA are useful tools to investigate the molecular mechanisms of viruses. The COVID‐19 pandemic prompted the development of several reverse genetics systems for SARS‐CoV‐2. The circular polymerase extension reaction (CPER) method enables the rapid generation of recombinant SARS‐CoV‐2; however, such PCR‐based approaches could introduce unwanted mutations due to PCR errors. Methods: To compare the accuracy of CPER and a classic reverse genetics method using bacterial artificial chromosome (BAC), SARS‐CoV‐2 Wuhan/Hu‐1/2019 was generated five times using BAC and five times using CPER. These 10 independent virus stocks were then deep sequencing, and the number of substitutions for which the frequency was greater than 10% was counted. Results: No nucleotide substitutions with a frequency of greater than 10% were observed in all five independent virus stocks generated by the BAC method. In contrast, three to five unwanted nucleotide substitutions with a frequency of more than 10% were detected in four of the five virus stocks generated by the CPER. Furthermore, four substitutions with frequencies greater than 20% were generated in three virus stocks by using the CPER. Conclusions: We found that the accuracy of the CPER method is lower than that of the BAC method. Our findings suggest care should be used when employing the CPER method. [ABSTRACT FROM AUTHOR]

Herpesviruses are major pathogens that infect humans and animals. Manipulating the large genome is critical for exploring the function of specific genes and studying the pathogenesis of herpesviruses and developing novel anti-viral vaccines and therapeutics. Bacterial artificial chromosome (BAC) technology significantly advanced the capacity of herpesviruses researchers to manipulate the virus genomes. In the past years, advancements in BAC-based genome manipulating and screening strategies of recombinant BACs have been achieved, which has promoted the study of the herpes virus. This review summarizes the advances in BAC-based gene editing technology and selection strategies. The merits and drawbacks of BAC-based herpesvirus genome editing methods and the application of BAC-based genome manipulation in viral research are also discussed. This review provides references relevant for researchers in selecting gene editing methods in herpes virus research. Despite the achievements in the genome manipulation of the herpes viruses, the efficiency of BAC-based genome manipulation is still not satisfactory. This review also highlights the need for developing more efficient genome-manipulating methods for herpes viruses. [ABSTRACT FROM AUTHOR]

The growth hormone (GH) locus has experienced a dramatic evolution in primates, becoming multigenic and diverse in anthropoids. Despite sequence information from a vast number of primate species, it has remained unclear how the multigene family was favored. We compared the structure and composition of apes' GH loci as a prerequisite to understanding their origin and possible evolutionary role. These thorough analyses of the GH loci of the chimpanzee, gorilla, and orangutan were done by resorting to previously sequenced bacterial artificial chromosomes (BACs) harboring them, as well as to their respective genome projects data available in GenBank. The GH loci of modern man, Neanderthal, gibbon, and wild boar were retrieved from GenBank. Coding regions, regulatory elements, and repetitive sequences were identified and compared among species. The GH loci of all the analyzed species are flanked by the genes CD79B (5′) and ICAM-1 (3′). In man, Neanderthal, and chimpanzee, the loci were integrated by five almost indistinguishable genes; however, in the former two, they rendered three different hormones, and in the latter, four different proteins were derived. Gorilla exhibited six genes, gibbon seven, and orangutan four. The sequences of the proximal promoters, enhancers, P-elements, and a locus control region (LCR) were highly conserved. The locus evolution might have implicated duplications of the ancestral pituitary gene (GH-N) and subsequent diversification of the copies, leading to the placental single GH-V gene and the multiple CSH genes. [ABSTRACT FROM AUTHOR]

Objective: The mechanistic target of rapamycin (mTOR) kinase is one of the master coordinators of cellular stress responses, regulating metabolism, autophagy, and apoptosis. We recently reported that staufen1 (STAU1), a stress granule (SG) protein, was overabundant in fibroblast cell lines from patients with spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis, frontotemporal degeneration, Huntington's, Alzheimer's, and Parkinson's diseases as well as animal models, and patient tissues. STAU1 overabundance is associated with mTOR hyperactivation and links SG formation with autophagy. Our objective was to determine the mechanism of mTOR regulation by STAU1. Methods: We determined STAU1 abundance with disease‐ and chemical‐induced cellular stressors in patient cells and animal models. We also used RNA‐binding assays to contextualize STAU1 interaction with MTOR mRNA. Results: STAU1 and mTOR were overabundant in bacterial artificial chromosome (BAC)‐C9ORF72, ATXN2Q127, and Thy1‐TDP‐43 transgenic mouse models. Reducing STAU1 levels in these mice normalized mTOR levels and activity and autophagy‐related marker proteins. We also saw increased STAU1 levels in HEK293 cells transfected to express C9ORF72‐relevant dipeptide repeats (DPRs). Conversely, DPR accumulations were not observed in cells treated by STAU1 RNA interference (RNAi). Overexpression of STAU1 in HEK293 cells increased mTOR levels through direct MTOR mRNA interaction, activating downstream targets and impairing autophagic flux. Targeting mTOR by rapamycin or RNAi normalized STAU1 abundance in an SCA2 cellular model. Interpretation: STAU1 interaction with mTOR drives its hyperactivation and inhibits autophagic flux in multiple models of neurodegeneration. Staufen, therefore, constitutes a novel target to modulate mTOR activity and autophagy, and for the treatment of neurodegenerative diseases. ANN NEUROL 2023;93:398–416 [ABSTRACT FROM AUTHOR]

Endometrial cancer (EC) is the most common gynaecological malignancy with increasing incidence in developed countries. As gold standard, hysteroscopy confirms only 30% of suspected ECs. The detection of EC cells in the vagina by fluorescence in situ hybridization (FISH) after a smear test could reduce invasive procedures in the future. Using array‐based comparative genome hybridization (aCGH) on 65 endometrial carcinomas, most frequently imbalanced regions of the tumour genome were identified. Bacterial artificial chromosomes were used to generate FISH‐probes homologue to these human regions. The FISH test was hybridized on swabs specimens collected from the vaginal cavity. Samples from six patients without EC were selected as a negative control and on 13 patients with known EC as a positive control. To distinguish between benign and EC cases, the cut‐off value has been defined. A first validation of this EC‐FISH Test was performed with swabs from 41 patients with suspected EC. The most common genomic imbalances in EC are around the CTNNB1, FBXW7 and APC genes. The cut‐off is defined at 32% of analysed cells without diploid signal pattern. This differs significantly between the positive and negative controls (p < 0.001). In a first validation cohort of 41 patients with suspected EC, the EC‐FISH Test distinguishes patients with and without EC with a sensitivity of 91% and a specificity of 83%. The negative predictive value is 96%. This is the first report of a non‐invasive EC‐FISH Test to predict EC in women with suspected EC. [ABSTRACT FROM AUTHOR]

Background: A historically definitive marker for cholinergic neurons is choline acetyltransferase (ChAT), a synthesizing enzyme for acetylcholine, (ACh), which can be found in high concentrations in cholinergic neurons, both in the central and peripheral nervous systems. ChAT, is produced in the body of the neuron, transported to the nerve terminal (where its concentration is highest), and catalyzes the transfer of an acetyl group from the coenzyme acetyl-CoA to choline, yielding ACh. The creation of bacterial artificial chromosome (BAC) transgenic mice that express promoter-specific fluorescent reporter proteins (green fluorescent protein—[GFP]) provided an enormous advantage for neuroscience. Both in vivo and in vitro experimental methods benefited from the transgenic visualization of cholinergic neurons. Mice were created by adding a BAC clone into the ChAT locus, in which enhanced GFP (eGFP) is inserted into exon 3 at the ChAT initiation codon, robustly and supposedly selectively expressing eGFP in all cholinergic neurons and fibers in the central and peripheral nervous systems as well as in non-neuronal cells. Methods: This project systematically compared the exact distribution of the ChAT-eGFP expressing neurons in the brain with the expression of ChAT by immunohistochemistry using mapping and also made comparisons with in situ hybridization (ISH). Results: We qualitatively described the distribution of ChAT-eGFP neurons in the mouse brain by comparing it with the distribution of immunoreactive neurons and ISH data, paying special attention to areas where the expression did not overlap, such as the cortex, striatum, thalamus and hypothalamus. We found a complete overlap between the transgenic expression of eGFP and the immunohistochemical staining in the areas of the cholinergic basal forebrain. However, in the cortex and hippocampus, we found small neurons that were only labeled with the antibody and not expressed eGFP or vice versa. Most importantly, we found no transgenic expression of eGFP in the lateral dorsal, ventral and dorsomedial tegmental nuclei cholinergic cells. Conclusion: While the majority of the forebrain ChAT expression was aligned in the transgenic animals with immunohistochemistry, other areas of interest, such as the brainstem should be considered before choosing this particular transgenic mouse line. [ABSTRACT FROM AUTHOR]

Genetic approaches that allow lineage tracing are essential to our future understanding of melanocytes and melanoma. To date, the approaches used to label melanocytes in mice have relied on random integration of transgenes driven by the promoters of the Tyrosinase and Dopachrome tautomerase genes, knock‐in to the Dopachrome tautomerase locus or knock‐in to the Mlana locus in a bacterial artificial chromosome. These strategies result in expression in other tissues such as telencephalon and other cell types such as nerves. Here we used homologous recombination in mouse embryonic stem cells to generate a targeted multicistronic allele of the Pmel locus that drives melanocyte‐specific expression of CreERT2, nuclear localised H2B‐Cerulean and membrane localised marcks‐mKate2 allowing live imaging of melanocytes and activation of other conditional alleles. We combined this allele with R26R‐EYFP mice allowing induction of EYFP expression on administration of tamoxifen or its metabolite 4‐OHT. The fluorescent proteins H2B‐Cerulean and marcks‐mKate2 label the cell nucleus and plasma membrane respectively allowing live imaging and FACS isolation of melanoblasts and melanocytes as well as serving to provide an internal control allowing estimation of recombination efficiency after administration of tamoxifen. We demonstrate the utility of the transgene in embryonic and adult tissues. [ABSTRACT FROM AUTHOR]

Introduction: This study explored the application of bacterial artificial chromosomes (BACs)-on-Beads (BoBs) technique, especially its ability to detect microdeletion/microduplication regions with a single probe. Methods: Both chromosome karyotyping and BoBs technique were applied on a total of 2218 pregnant women. Chromosome microarray analysis (CMA) was performed on patients whose cells were reported as being abnormal by BoBs technique with a single probe. Results: Twenty-two cases were detected as microdeletion/microduplication with a single probe, which was consistent with the CMA results. Conclusions: We believe that the microdeletion/microduplication results detected by BoBs technique with a single probe provide comprehensive guidance for prenatal diagnosis. [ABSTRACT FROM AUTHOR]

Equine herpesvirus 1 (EHV1) infection is a global health problem in equines and the virus is responsible for abortions, respiratory disease and myeloencephalitis in horses. Disease management requires proper biosecurity and immunoprophylactic measures. Vaccines strengthening both arms of immunity are essential for proper control and there has been a continuous focus in this area for generation of better vaccines. Here we report construction of bacterial artificial chromosome (BAC) clone of EHV-1 strain Tohana for mutagenesis of the virus and generation of gE gene deletion mutant EHV1. The BAC clone was generated by inserting the mini-F plasmid replacing ORF71 of EHV1 and transforming into E. coli for generation of EHV1-BAC. The infectious virus was regenerated from EHV-1 BAC DNA in RK13 cells. To check utility of EHV1-BAC, we have generated mutant EHV1 by deleting the virulence-associated gE gene. The mutant virus (vToHΔgE) showed significantly reduced plaque size without affecting replication efficiency. Pathological evaluation of lesions in BALB/c mice infected with vToHΔgE revealed reduction in clinical signs and pathology in comparison to the wild-type virus. Generation of infectious BAC of EHV1 and its usage in construction of attenuated viruses shows potential of the technology for development of indigenous modified live vaccine for EHV1. [ABSTRACT FROM AUTHOR]

Construction of a physical chromosome map of a species is important for positional cloning, targeted marker development, fine mapping of genes, selection of candidate genes for molecular breeding, as well as understanding the genome organization. The genomic libraries in the form of bacterial artificial chromosome (BAC) clones are also a very useful resource for physical mapping and identification and isolation of full-length genes and the related cis acting elements. Some BAC-FISH based studies reported in the past were gene based physical chromosome maps of Clarias magur (magur) to understand the genome organization of the species and to establish the relationships with other species in respect to genes' organization and evolution in the past. In the present study, we generated end sequences of the BAC clones and analyzed those end sequences within the scaffolds of the draft genome of magur to identify and map the genes bioinformatically for each clone. A total of 36 clones mostly possessing genes were identified and used in probe construction and their subsequent hybridization on the metaphase chromosomes of magur. This study successfully mapped all 36 specific clones on 16 chromosome pairs, out of 25 pairs of magur chromosomes. These clones are now recognized as chromosome-specific makers, which are an aid in individual chromosome identification and fine assembly of the genome sequence, and will ultimately help in developing anchored genes' map on the chromosomes of C. magur for understanding their organization, inheritance of important fishery traits and evolution of magur with respect to channel catfish, zebrafish and other species. [ABSTRACT FROM AUTHOR]

Simple Summary: Numerous tiny (micro)chromosomes are a characteristic feature associated with birds, being found in smaller numbers in other organisms and absent in many, such as mammals. Although microchromosomes constitute a large portion of the genome in birds, data on them pertaining to comparative studies between birds are still scarce. This is the case in shorebirds (Charadriiformes), a group with a great variety of species. The aim of this study was to provide insight regarding the evolution of the microchromosomes of three species of shorebirds—the red knot (Calidris canutus), the wattled jacana (Jacana jacana), and the southern lapwing (Vanellus chilensis). The experiments are referred to as cross-species fluorescence in situ hybridization (FISH) mapping using probes called bacterial artificial chromosomes (or BACs), two (one labelled in red and one labelled in green) for every microchromosome. The results thus appear as the microchrochromosome with one green and one red end, revealing different patterns of organization over evolutionary time. In the red knot, they fuse together, but in the southern lapwing, they hardly change. We also described a new chromosome number for the red knot (92 in total). In conclusion, this study contributed to the understanding of microchromosomes organization and evolution of three shorebird species. Microchromosomes, once considered unimportant elements of the genome, represent fundamental building blocks of bird karyotypes. Shorebirds (Charadriiformes) comprise a wide variety of approximately 390 species and are considered a valuable model group for biological studies. Despite this variety, cytogenetic analysis is still very scarce in this bird order. Thus, the aim of this study was to provide insight into the Charadriiformes karyotype, with emphasis on microchromosome evolution in three species of shorebirds—Calidris canutus, Jacana jacana, and Vanellus chilensis—combining classical and molecular approaches. Cross-species FISH mapping applied two BAC probes for each microchromosome, GGA10–28 (except GGA16). The experiments revealed different patterns of microchromosome organization in the species investigated. Hence, while in C. canutus, we found two microchromosomes involved in chromosome fusions, they were present as single pairs in V. chilensis. We also described a new chromosome number for C. canutus (2n = 92). Hence, this study contributed to the understanding of genome organization and evolution of three shorebird species. [ABSTRACT FROM AUTHOR]

Pyramiding different fire blight resistance genes and QTLs in future apple cultivars is the most eco-friendly way to combat this disease. Identification of strong fire blight resistance donors, and introgression of their resistance into apple breeding material are a continuing effort of breeding programs. Thus, enormous effort is been put into breeding research to understand host – pathogen interactions and mechanisms of resistance found in Malus. The crabapple Malus fusca (accession MAL0045) is highly resistant to fire blight, and although resistance is strain-dependent, resistance of MAL0045 is not overcome by any known strain of Erwinia amylovora to date. A strong fire blight resistance locus (FB_Mfu10) was fine mapped to an interval of 0.33 Centimorgan (cM) on linkage group (LG) 10 of MAL0045 using 1888 progenies. Subsequently, a single bacterial artificial chromosome (BAC) clone (46H22), which harbours FB_Mfu10-resistance alleles, was identified in a MAL0045 BAC library and sequenced using MiSeq illumina leading to the assembly of 45 contigs. Analyses of the sequence of 46H22 led to the identification of a receptor-like kinase candidate gene. Here, we report about resequencing 46H22 using MinION Oxford Nanopore and successfully assembled the sequences into a single contig, which allowed for identifying additional candidate genes. [ABSTRACT FROM AUTHOR]

For the past three decades, the porcine epidemic diarrhea virus (PEDV) has remained an enormous threat to the South Korean swine industry. The scarcity of an effective method for manipulating viral genomes has impeded research progress in PEDV biology and vaccinology. Here, we report the development of reverse genetics systems using two novel infectious full-length cDNA clones of a Korean highly pathogenic-G2b strain, KNU-141112, and its live attenuated vaccine strain, S DEL5/ORF3, in a bacterial artificial chromosome (BAC) under the control of a eukaryotic promoter. Direct transfection of cells with each recombinant BAC clone induced cytopathic effects and produced infectious progeny. The reconstituted viruses, icKNU-141112 and icS DEL5/ORF3, harboring genetic markers, displayed phenotypic and genotypic properties identical to their respective parental viruses. Using the DNA-launched KNU-141112 infectious cDNA clone as a backbone, two types of recombinant viruses were generated. First, we edited the open reading frame 3 (ORF3) gene, as cell-adapted strains lose full-length ORF3, and replaced this region with an enhanced green fluorescent protein (EGFP) gene to generate icPEDV-EGFP. This mutant virus presented parental virus-like growth kinetics and stably retained robust EGFP expression, indicating that ORF3 is dispensable for PEDV replication in cell culture and is a tolerant location for exogeneous gene acceptance. However, the plaque size and syncytia phenotypes of ORF3-null icPEDV-EGFP were larger than those of icKNU-141112 but similar to ORF3-null icS DEL5/ORF3, suggesting a potential role of ORF3 in PEDV cytopathology. Second, we substituted the spike (S) gene with a heterologous S protein, designated S51, from a variant of interest (VOI), which was the most genetically and phylogenetically distant from KNU-141112. The infectious recombinant VOI, named icPEDV-S51, could be recovered, and the rescued virus showed indistinguishable growth characteristics compared to icKNU-141112. Virus cross-neutralization and structural analyses revealed antigenic differences in S between icKNU-141112 and icPEDV-S51, suggesting that genetic and conformational changes mapped within the neutralizing epitopes of S51 could impair the neutralization capacity and cause considerable immune evasion. Collectively, while the established molecular clones afford convenient, versatile platforms for PEDV genome manipulation, allowing for corroborating the molecular basis of viral replication and pathogenesis, they also provide key infrastructural frameworks for developing new vaccines and coronaviral vectors. [ABSTRACT FROM AUTHOR]

Background: HAP1, a near-haploid human leukemic cancer cell line is often used in combination with CRISPR-Cas9 gene editing technology for genetic screens. HAP1 carries the Philadelphia chromosome (Ph) and an additional ~ 30 Mb fragment of chromosome 15 inserted into chromosome 19. The potential use of an in vitro cell line as a model system in biomedical research studies depends on its ability to maintain genome stability. Being a cancer cell line with a near-haploid genome, HAP1 is prone to genetic instability, which is further compounded by its tendency to diploidise in culture spontaneously. Moreover, CRISPR-Cas9 gene editing coupled with prolonged in-vitro cell culturing has the potential to induce unintended 'off-target' cytogenetic mutations. To gain an insight into chromosomal instability (CIN) and karyotype heterogeneity, 19 HAP1 cell lines were cytogenetically characterised, 17 of which were near-haploids and two double-haploids, using multiplex fluorescence in situ hybridisation (M-FISH), at single cell resolution. We focused on novel numerical (N) and structural (S) CIN and discussed the potential causal factors for the observed instability. For each cell line we examined its ploidy, gene editing status and its length of in-vitro cell culturing. Results: Sixteen of the 19 cell lines had been gene edited with passage numbers ranging from 10 to 35. Diploidisation in 17 near-haploid cell lines ranged from 4 to 35% and percentage of N- and S-CIN in [1n] and [2n] metaphases ranged from 7 to 50% with two cell lines showing no CIN. Percentage of cells with CIN in the two double-haploid cell lines were 96% and 100% respectively. The most common S-CIN observed was deletion followed by translocation of both types, non-reciprocal and Robertsonian. Interestingly, we observed a prevalence of S-CIN associated with chromosome 13 in both near-and double-haploid cell lines, with a high incidence of Robertsonian translocation involving chromosome 13. Furthermore, locus-specific BAC (bacterial artificial chromosome) FISH enabled us to show for the first time that the additional chromosome 15 fragment is inserted into the p-arm rather than the q-arm of chromosome 19 of the HAP1 genome. Conclusion: Our study revealed a high incidence of CIN leading to karyotype heterogeneity in majority of the HAP1 cell lines with the number of chromosomal aberrations varying between cell lines. A noteworthy observation was the high frequency of structural chromosomal aberrations associated with chromosome 13. We showed that CRISPR-Cas9 gene editing technology in combination with spontaneous diploidisation and prolonged in-vitro cell culturing is potentially instrumental in inducing further chromosomal rearrangements in the HAP1 cell lines with existing CIN. We highlight the importance of maintaining cell lines at low passage and the need for regular monitoring to prevent implications in downstream applications. Our study also established that the additional fragment of chromosome 15 in the HAP1 genome is inserted into chromosome 19p rather than 19q. [ABSTRACT FROM AUTHOR]

Some of the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are less susceptible to neutralization with post-vaccine sera and monoclonal antibodies targeting the viral spike glycoprotein. This raises concerns of disease control, transmissibility, and severity. Numerous substitutions have been identified to increase viral fitness within the nucleocapsid and nonstructural proteins, in addition to spike mutations. Therefore, we sought to generate infectious viruses carrying only the variant-specific spike mutations in an identical backbone to evaluate the impact of spike and non-spike mutations in the virus life cycle. We used en passant mutagenesis to generate recombinant viruses carrying spike mutations of B.1 and B.1.617.2 variants using SARS-CoV-2- bacterial artificial chromosome (BAC). Neutralization assays using clinical sera yielded comparable results between recombinant viruses and corresponding clinical isolates. Non-spike mutations for both variants neither seemed to effect neutralization efficiencies with monoclonal antibodies nor the response to treatment with inhibitors. However, live-cell imaging and microscopy revealed differences, such as persisting syncytia and pronounced cytopathic effect formation, as well as their progression between BAC-derived viruses and clinical isolates in human lung epithelial cell lines and primary bronchial epithelial cells. Complementary RNA analyses further suggested a potential role of non-spike mutations in infection kinetics. [ABSTRACT FROM AUTHOR]