Search

β654-thalassemia is a prominent Chinese subtype of b-thalassemia, representing 17% of all cases of β-thalassemia in China. The molecular mechanism underlying this subtype involves the IVS-2-654 C→T mutation leading to aberrant β-globin RNA splicing. This results in an additional 73-nucleotide exon between exons 2 and 3 and leads to a severe thalassemia syndrome. Herein, we explored a CRISPR/Cas9 genome editing approach to eliminate the additional 73- nucleotide by targeting both the IVS-2-654 C→T and a cryptic acceptor splice site at IVS-2-579 in order to correct aberrant b-globin RNA splicing and ameliorate the clinical β-thalassemia syndrome in β654 mice. Gene-edited mice were generated by microinjection of sgRNA and Cas9 mRNA into one-cell embryos of β654 or control mice: 83.3% of live-born mice were gene-edited, 70% of which produced correctly spliced RNA. No off-target events were observed. The clinical symptoms, including hematologic parameters and tissue pathology of all of the edited β654 founders and their offspring were significantly improved compared to those of the non-edited β654 mice, consistent with the restoration of wild-type b-globin RNA expression. Notably, the survival rate of gene-edited heterozygous β654 mice increased significantly, and liveborn homozygous β654 mice were observed. Our study demonstrated a new and effective gene-editing approach that may provide groundwork for the exploration of β654-thalassemia therapy in the future.

Silt has the characteristics of obvious capillary water effect and strong water sensitivity. The flooding of the Yellow River caused the water level in Kaifeng to be high, and the damage of capillary water to the silt site of Kaifeng Zhouqiao site is increasing day by day. In order to improve the waterproof performance of the site soil, three kinds of silicone waterproof materials were selected, and the site soil was improved by internal mixing. The improvement effects of different materials were compared through the capillary water rise test, and the contact angle of the modified soil with the optimal ratio was measured. Microscopic tests were conducted to evaluate its wetting properties and reveal the mechanism of action of the modified materials. The results show that the three kinds of silicone waterproofing agents can improve the waterproofing effect of soil, among which 0.5% sodium methylsilicate modified soil has the most significant waterproofing effect; its capillary water absorption inhibition effect can reach 98.34%, and the contact angle is 137.06. The surface of the modified soil is hydrophobic after the addition of sodium methylsilicate. An evenly distributed waterproof film is thus formed on the surface of the soil particles, which changes the contact mode between the soil particles and strengthens the connection between the soil particles, so that the proportion of large pores decreased. The proportion of mesopores and small pores increased, which made the soil sample more compact. These results explain the improvement of the waterproof performance of the soil sample.

Natural hydraulic lime soil has good mechanical properties; as an earthen ruin restoration material, its durability is insufficient. Despite natural hydraulic lime being a topic that has been studied for several years from different researchers, it has not yet been fully considered for the improvement of durability. This work aims to experimentally investigate the enhancement of the durability properties of hydraulic lime-based. The performance of natural hydraulic limestone by adding sodium methyl silicate and organic silicon is examined and the effect of adding sodium methyl silicate on its performance and microstructure is studied. The 6%, 10%, and 15% lime–soil comparison test blocks of sodium silicate were compared with different lime–soil comparison test blocks not mixed with sodium methyl silicate; in addition to compression resistance, shear resistance, water absorption, and erosion resistance, dry–wet cycles were carried out, as well as microstructure testing and analysis. The results show that the addition of sodium methyl silicate enhances the compressive strength of hydraulic lime-modified soil, reduces its saturated water absorption, reduces its shear strength, improves its resistance to dry and wet cycles, and forms on the surface of the modified soil particles. The hydrophobic layer further improves its erosion resistance and water resistance. When the sodium methyl silicate content is 0.3%, the natural hydraulic lime soil has good mechanical properties and good durability, which is the optimal ratio.

Aiming at the path planning and decision-making problem, multi-automated guided vehicles (AGVs) have played an increasingly important role in the multi-stage industries, e.g., textile spinning. We recast a framework to investigate the improved genetic algorithm (GA) on multi-AGV path optimization within spinning drawing frames to solve the complex multi-AGV maneuvering scheduling decision and path planning problem. The study reported in this paper simplifies the scheduling model to meet the drawing workshop's real-time application requirements. According to the characteristics of decision variables, the model divides into two decision variables: time-independent variables and time-dependent variables. The first step is to use a GA to solve the AGV resource allocation problem based on the AGV resource pool strategy and specify the sliver can's transportation task. The second step is to determine the AGV transportation scheduling problem based on the sliver can-AGV matching information obtained in the first step. One significant advantage of the presented approach is that the fitness function is calculated based on the machine selection strategy, AGV resource pool strategy, and the process constraints, determining the scheduling sequence of the AGVs to deliver can. Moreover, it discovered that double-path decision-making constraints minimize the total path distance of all AGVs, and minimizing single-path distances of each AGVs exerted. By using the improved GA, simulation results show that the total path distance was shortened.

SUMMARY The lung is the primary site of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced immunopathology whereby the virus enters the host cells by binding to angiotensin-converting enzyme 2 (ACE2). Sophisticated regeneration and repair programs exist in the lungs to replenish injured cell populations. However, known resident stem/progenitor cells have been demonstrated to express ACE2, raising a substantial concern regarding the long-term consequences of impaired lung regeneration after SARS-CoV-2 infection. Moreover, clinical treatments may also affect lung repair from antiviral drug candidates to mechanical ventilation. In this review, we highlight how SARS-CoV-2 disrupts a program that governs lung homeostasis. We also summarize the current efforts of targeted therapy and supportive treatments for COVID-19 patients. In addition, we discuss the pros and cons of cell therapy with mesenchymal stem cells or resident lung epithelial stem/progenitor cells in preventing post-acute sequelae of COVID-19. We propose that, in addition to symptomatic treatments being developed and applied in the clinic, targeting lung regeneration is also essential to restore lung homeostasis in COVID-19 patients.

Induced pluripotent stem cells (iPSCs), generated from somatic cells, not only possess similar characteristics with embryonic stem cells (ESCs), but also present more advantages than ESCs in medical applications. The classical induction method that utilizes the integration of exogenous genes into chromosomes may raise the potential risk of the safety of iPSCs. To investigate the potential correlation between the integration sites of exogenous transcription factors (TFs) and iPSCs’ pluripotency and safety, the integration of exogenous genes in three iPSC lines, which met the golden standard of murine developmental assay (tetraploid complementation), were analyzed. Twenty-two integration sites of exogenous TFs were identified by nested inverse polymerase chain reaction (iPCR) and 39 flanking genes’ functions were analyzed by gene ontology (GO). In the 22 integrated sites, 17 (77.3%) were located in the intergenic regions and the remainder were located in introns far from the transcription start sites. Microarray analysis of the flanking genes in these cells showed that there was no distinct difference in expression levels between the iPSCs, ESCs and mouse embryonic fibroblast (MEF), suggesting that the integration of exogenous TFs has no significant influence on the expression of flanking genes. Gene ontology analysis showed that although most of the flanking genes were housekeeping genes, which were necessary for basic life activity, none of these 39 flanking genes have correlation with tumorigenesis or embryogenesis, suggesting that the integration sites hold low risk of tumorigenesis.

This article investigates the relationship between the tracking performance and coupling effect for attitude tracking issue of hypersonic reentry vehicle (HRV), and proposes a performance-involved coupling effect-triggered robust control scheme. The main feature of the article lies in that the influence of the coupling effect on the performance is discussed via a defined performance-involved coupling effect indicator. Consequently, the proposed control ensures the transient performance within the prescribed limitation in all phases, and the better transient performance is achieved with the help of the sliding mode control and disturbance observer technique enhancing the robustness. Comparison simulations and Monte Carlo tests under coefficients perturbation on the HRV system demonstrate the effectiveness of the proposed control strategy.

The prevalence of staphylococcal infection and the emergence of multidrug resistance of Staphylococcus aureus (S. aureus) are major concerns in food safety and public health. This study aimed to investigate the prevalence of S. aureus isolated from traditional Chinese Rubing and Rushan cheese, antimicrobial resistance profiles, genomic characteristics, and predict antimicrobial resistance genes (ARGs). From 124 samples, 18 of 62 (29.03%) of Rubing and 5 of 62 (8.06%) of Rushan cheese were confirmed to be S. aureus positive by standard culture-based methods. Twenty-three coagulase-positive staphylococci isolates were grouped into 16 clusters by pulsed-field gel electrophoresis and subjected to routine susceptibility testing to 12 antibiotics. Those isolates exhibited high resistance to penicillin (100%), erythromycin, trimethoprim-sulphamethoxazole (34.78%), oxacillin, clindamycin, and cefoxitin (21.74%). Multidrug-resistant (MDR) S. aureus was found in 34.78% (8 of 23) of isolates. Further, S. aureus strain DC.RB_015 isolated from Rubing cheese, recognized as the most resistant to six antibiotics, was selected for whole-genome sequencing (WGS), continued with in silico approaches. S. aureus DC.RB_015 had a single chromosome size of 2,794,578 bp and a plasmid size of 22,961 bp. The strain harbored 18 predicted ARGs, including eight efflux pump genes (mepA, tet(K), arlR, arlS, norA, mgrA, tet(38), LmrS), one peptidoglycan biosynthesis gene (bacA), two β-lactams resistance genes (mecA, blaZ), and seven genes conferring other antimicrobial resistance (APH(3′)-IIIa, aad(6), ErmB, SAT-4, mecR1, GlpT, murA). The results of this study expand the knowledge of S. aureus strain DC.RB_015, increase food safety awareness, and will be helpful in establishing therapeutic therapy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03072-4.

Kaifeng Zhouqiao site is located in the traffic trunk line in Gulou District of Kaifeng City. The dynamic load of urban traffic is large. In addition, the silt has poor stability of its particle skeleton structure, large porosity and poor mechanical performance. Under the action of dynamic load, the soil of the site will suffer from cracking, collapse, unstable deformation and overall stability damage. In order to enhance the stability of the soil, this paper uses sodium methylsilicate and lignin fiber to modify the site soil, evaluates the mechanical properties of the improved soil through the compression and shear tests, evaluates the durability of the improved soil through the dry wet cycle test, and reveals its modification mechanism through the micro experiment, so that the mechanical properties of the site soil can be improved, so as to achieve the purpose of repair and reinforcement. The experiment shows that the effect of improving the compressive strength of soil is the best when the content of sodium methylsilicate is 0.3%–0.5%, and the effect of improving the shear strength of soil is the best when the content of lignin is 0.5%–2%. The maximum mass loss rate of the composite modified sample after 10 dry and wet cycles is only 0.71%. The comprehensive analysis determines that the best proportion of the composite is 0.5% sodium methylsilicate and 2% lignin fiber. The modified soil has good waterproof and mechanical properties.

This article takes the stone statues of the Northern Song Dynasty imperial mausoleum as the research object. Aiming at the characteristics of the stone statues low weight and small building area, combined with the serious weathering of the stone statues, the reasons for the inclination and the defects of the traditional round soil excavation holes, an elliptical soil excavation correction method is proposed. Adopt the method of combining numerical simulation and experiment to carry out the research of elliptical excavation and tilt correction of stone statues. Research indicates: The settlement difference between the excavation side and the non-excavation side increases with the increase of the span-to-height ratio of the excavation hole; The shallower the depth of the digging hole, the greater the settlement difference between the digging side and the non-digging side; The settlement difference between the digging side and the non-digging side decreases with the increase of the hole spacing; The settlement of the superstructure mainly occurs within 4 to 5 hours after the soil is excavated, and then enters a slow growth stage, and finally gradually stabilizes; During the test, the failure of the digging hole belongs to plastic failure. The damage of the digging hole is firstly destroyed from both sides of the hole, and the upper part of the hole is destroyed immediately, and the upper structure edge is destroyed first.

In this paper, a finite-time control strategy based on back-stepping method combining with a terminal sliding mode control (TSMC) and a nonlinear disturbance observer (NDO) is proposed for the longitudinal dynamic model of hypersonic vehicle (HV). Firstly, the model of HV is transformed into two strict feedback subsystem: the mismatched subsystem of altitude and the matched subsystem of velocity. Then, the TSMC and back-stepping method is incorporated to cope with the unmatched issue in the HV altitude subsystem. In addition, a NDO based on a finite-time-convergent differentiator (FD) is proposed to estimate the lumped disturbances. The finite-time stability condition of the system is established via the Lyapunov theory. Finally, the robustness and effectiveness of the method are verified by simulations.

β654-thalassemia is a prominent Chinese subtype of b-thalassemia, representing 17% of all cases of β-thalassemia in China. The molecular mechanism underlying this subtype involves the IVS-2-654 C→T mutation leading to aberrant β-globin RNA splicing. This results in an additional 73-nucleotide exon between exons 2 and 3 and leads to a severe thalassemia syndrome. Herein, we explored a CRISPR/Cas9 genome editing approach to eliminate the additional 73- nucleotide by targeting both the IVS-2-654 C→T and a cryptic acceptor splice site at IVS-2-579 in order to correct aberrant b-globin RNA splicing and ameliorate the clinical β-thalassemia syndrome in β654 mice. Gene-edited mice were generated by microinjection of sgRNA and Cas9 mRNA into one-cell embryos of β654 or control mice: 83.3% of live-born mice were gene-edited, 70% of which produced correctly spliced RNA. No off-target events were observed. The clinical symptoms, including hematologic parameters and tissue pathology of all of the edited β654 founders and their offspring were significantly improved compared to those of the non-edited β654 mice, consistent with the restoration of wild-type b-globin RNA expression. Notably, the survival rate of gene-edited heterozygous β654 mice increased significantly, and liveborn homozygous β654 mice were observed. Our study demonstrated a new and effective gene-editing approach that may provide groundwork for the exploration of β654-thalassemia therapy in the future.

Pointed at a problem that leads to the high complexity of the production management tasks in the multi-stage spinning industry, mixed flow batch production is often the case in response to a customer&rsquo, s personalized demands. Manual handling cans have a large number of tasks, and there is a long turnover period in their semi-finished products. A novel heuristic research was conducted that considered mixed-flow shop scheduling problems with automated guided vehicle (AGV) distribution and path planning to prevent conflict and deadlock by optimizing distribution efficiency and improving the automation degree of can distribution in a draw-out workshop. In this paper, a cross-region shared resource pool and an inter-regional independent resource pool, two AGV predictive scheduling strategies are established for the ring-spinning combing process. Besides completion time, AGV utilization rate and unit AGV time also analyzed with the bottleneck process of the production line. The results of the optimal computational experiment prove that a draw frame equipped with multi-AGV and coordinated scheduling optimization will significantly improve the efficiency of can distribution. Flow-shop predictive modeling for multi-AGV resources is scarce in the literature, even though this modeling also produces, for each AGV, a control mode and, if essential, a preventive maintenance plan.

Conjugated linoleic acid (CLA) is an unsaturated fatty acid with many active functions. Here, we demonstrate quorum sensing (QS) to study mechanisms involved in CLA production of L. fermentum L1. Addition of linoleic acid (LA) with increased concentration results in promising CLA production of 2.22–10.36 mg/mL, cell density and autoinducer-2 (AI-2) involved in QS of L1. Further, proteomics analysis quantified up-expression of 185 differentially expressed proteins (DEPs) upon addition substrate of 8% LA, including myosin cross-reactive antigen protein, enolase (eno), and S-ribosylhomocysteine lyase (luxS), while 174 DEPs were down-regulated. Furthermore, S-Adenosyl- l -methionine metabolic pathway involved in luxS/AI-2 QS system was up-regulated. Additionally, protein-protein interaction network leverage key proteins, luxS and eno responsible for CLA production. Finally, furanone inhibitor block luxS/AI-2 QS system to allow AI-2 content, CLA, and cell density of L1 to decrease level. Western blot analysis confirmed, furanone successfully inhibited expression of luxS and eno. Whereas, luxS/AI-2 QS system was activated with LA results in increase expression of eno and production of CLA. Collectively, this study provides a new mechanism of CLA production by lactic acid bacteria.

In this paper, the vibration effect of Longhai Railway on a granary ruins is taken as the research object. Firstly, the vibration response of the granary ruins is obtained by field test. Then, using FLAC3D finite element analysis software to establish an overall calculation model, and the shock absorption effect of vibration isolation barrier is analyzed and predicted. The conclusions are as follows: the horizontal vibration of granary ruins is stronger than the vertical vibration; because of the special structure of the granary ruins, when the vibration propagates to the interface of the cellar wall, it will produce horizontal vibration amplification phenomenon, and the vibration intensity of the granary ruins away from the vibration source will decrease faster; discontinuous concrete isolation piles are used to protect granary ruins. Under the premise of considering more economical, double-row piles with staggered layout have the best vibration isolation effect.

In order to accurately evaluate the stability of the present situation of the soil site, considering that the stability of the soil site is affected by its own structural characteristics and the external environment, the multiple factors affecting the stability of the soil site are determined, and the stability evaluation system of the soil site is constructed. The evaluation system is divided into three levels: target level, three primary levels and fifteen secondary levels, and four evaluation levels. Firstly, the weight value of the soil site stability evaluation index was determined by AHP, and the rationality of the evaluation index was verified by consistency test. Based on the expert experience and two kinds of ridge type membership functions, the fuzzy comprehensive evaluation model of soil site stability at three levels and two stages was established. Finally, combined with the analysis of engineering examples, it is shown that the fuzzy analytic hierarchy process is an effective method for the stability evaluation of soil sites, which can not only achieve the dual goals of safety, reliability and economic rationality, but also provide a basis for the selection of soil site disease control schemes.

In recent years, as a new type of ice storage medium, ice slurry has been widely applied in multiple fields. The scraped-surface method has remained the focus of researches, thanks to its advantages such as technical maturity, low cost and stable performance, etc. In this paper, the attributes of ice slurry was briefly introduced and then an analysis and comparison of several common ice slurry generation methods was presented with a summary of the advantages of the scraped-surface method. Then, the current research status of scraping-type ice slurry generation method was introduced. In the final place, this paper would summarizes the existing research fruition and propose a zigzag scraper, and future development and trend are looked forward to.

With the increasing demand for frozen food in China, the need for better cold chain logistics is also becoming more urgent, promoting the development of related new technologies. This paper focuses on the research of ice slurry and intelligent temperature control technology in cold chain logistics, expounds on the research background, present situation and innovation research, and finally discusses the problems existing in cold chain logistics technology and corresponding solutions.

Aquatic products are very ideal healthy food. Cold chain logistics is an effective way to ensure the quality of aquatic products in the circulation process. Zhoushan City (China) is situated in Zhoushan archipelago. Zhoushan fishing ground is the largest fishing ground in China, with abundant fishery resources. This paper expounds the theory of cold chain logistics, analyzes the present situation of cold chain logistics of Zhoushan aquatic products, points out the problems existing in cold chain logistics of Zhoushan aquatic products, and puts forward corresponding solutions, which has a positive role in promoting the development of cold chain logistics of Zhoushan aquatic products.

A new simple and efficient one-pot synthesis of 2-fluoroalkyl substituted indoles from 2-aminobenzyl alcohols with fluorine-containing carboxylic acids in the presence of Ph3P, CCl4, and NEt3 is described. Various kinds of 2-fluoroalkyl substituted indole derivatives can be conveniently prepared.

Streptomyces phage φC31 integrase induces efficient site-specific recombination capable of integrating exogenous genes at pseudo attP sites in human, mouse, rat, rabbit, sheep, Drosophila, and bovine genomes. However, the φC31-mediated recombination between attB and the corresponding pseudo attP sites has not been investigated in Capra hircus. Here, we identified eight pseudo attP sites located in the intron or intergenic regions of the C. hircus genome, and demonstrated different levels of foreign gene expression after φC31 integrase-mediated integration. These pseudo attP sites share similar sequences with each other and with pseudo attP sites in other mammalian genomes, and these are associated with a neighboring consensus motif found in other genomes. The application of the φC31 integrase system in C. hircus provides a new option for genetic engineering of this economically important goat species.

Based on several unpredictable reasons, buildings will have varying degrees of tilt damage. As for Line-Shaped buildings structure, since its own structure has a large aspect ratio, it is more prone to appear the tilt damage than other types of buildings. However, some already existed building correction methods are not suitable for line-shaped buildings. So the present study proposes and analyzes the correction method which is more effective to deal with the tilt damage of Line-Shaped buildings structure. Regarded the ancient the Screen Wall as an example, firstly the study analyzes the reasons of damage, and then puts up with corresponding methods. It can devote an available method for such projects for reference.

The introduction of double-strand breaks (DSBs) at target sites could greatly enhance homologous recombination, and engineered nucleases, such as zinc finger and transcription activator-like effector nucleases, have been successfully developed for making such breaks. In this study, we present a highly efficient site-specific integration strategy based on homologous recombination and ΦC31 integrase. An attB sequence was introduced at the homologous arm of an insertion targeting vector. DSBs at the target locus and donor were then simultaneously generated by the ΦC31 integrase when co-transfected with the donor vector, consequently stimulating homologous recombination. The results demonstrated that our strategy is feasible and the efficiency at the BF4 target site, which we previously identified in the bovine genome, was as high as 93%. The frequency at another site (BF10) was almost two-fold greater in comparison to the vector without homologous arms. This technology requires no sophisticated nuclease design efforts, and the off-target effect is reduced by ΦC31 integrase compared to the use of engineered nucleases, thereby offering a simple and safe way to effectively express a donor gene at a desired locus. This development has great potential value, especially in transgenesis or gene therapy applications.

Embryonic stem cells (ESCs) may be useful as a therapeutic source of cells for the production of healthy tissue; however, they are associated with certain challenges including immunorejection as well as ethical issues. Induced pluripotent stem cells (iPSCs) are a promising substitute since a patient's own adult cells would serve as tissue precursors. Ethical concerns prevent a full evaluation of the developmental potency of human ESCs and iPSCs, therefore, mouse iPSC models are required for protocol development and safety assessments. We used a modified culturing protocol to differentiate pluripotent cells from a mouse iPS cell line and two mouse ES cell lines into neurons. Our results indicated that all three pluripotent stem cell lines underwent nearly the same differentiation process when induced to form neurons in vitro. Genomic expression microarray profiling and single-cell RT-qPCR were used to analyze the neural lineage differentiation process, and more than one thousand differentially expressed genes involved in multiple molecular processes relevant to neural development were identified.

To explore the disease-causing mutations in a patient suspected for giant axonal neuropathy(GAN).Target sequence capture sequencing was used to screen potential mutations in genomic DNA extracted from peripheral blood sample of the patient. Sanger sequencing was applied to confirm the detected mutation. The mutation was verified among 400 GAN alleles from 200 healthy individuals by Sanger sequencing. The function of the mutations was predicted by bioinformatics analysis.The patient was identified as a compound heterozygote carrying two novel pathogenic GAN mutations, i.e., c.778GT (p.Glu260Ter) and c.277GA (p.Gly93Arg). Sanger sequencing confirmed that the c.778GT (p.Glu260Ter) mutation was inherited from his father, while c.277GA (p.Gly93Arg) was inherited from his mother. The same mutations was not found in the 200 healthy individuals. Bioinformatics analysis predicted that the two mutations probably caused functional abnormality of gigaxonin.Two novel GAN mutations were detected in a patient with GAN. Both mutations are pathogenic and can cause abnormalities of gigaxonin structure and function, leading to pathogenesis of GAN. The results may also offer valuable information for similar diseases.

A new simple and efficient one-pot synthesis of 2-fluoroalkyl substituted indoles from 2-aminobenzyl alcohols with fluorine-containing carboxylic acids in the presence of Ph3P, CCl4, and NEt3 is described. Various kinds of 2-fluoroalkyl substituted indole derivatives can be conveniently prepared.

ΦC31 integrase, a site-specific recombinase, can catalyze integration of circular DNA bearing attB site into pseudo attP sites in mammalian genomes. However, the integration efficiency mediated by integrase is relatively low. Our study centered on the investigation of the impact of the position, orientation, and number of attBs in the donor plasmid on the efficiency of ΦC31 integrase system. Donor plasmids bearing various types of attBs (including forward and reverse directions, tandem, and intersperse) and reporter enhanced green fluorescent protein (EGFP) were constructed. The plasmids plus helper plasmid encoding integrase were co-transfected into HeLa cells. After G418 selection, the resistant cell colonies were counted for calculating chromosomal integration frequency. EGFP expression was detected by fluorescence-activated cell sorter and enzyme-linked immunosorbent assay analysis. The results showed that efficiency of integration mediated by integrase accounted for 70% ± 7.1% of total integration events in the transfected HeLa cells. Compared with a forward orientation of attB in donor plasmid, a reverse direction of attB or interspersed attBs showed 1.5- or 2.8-fold increase in integration efficiency, respectively, while tandem attBs in donor plasmids caused a decreased efficiency of integration. We conclude that the adjustment of attB sites in donor plasmids may be of value for gene therapy and routine genetic engineering by using ΦC31 integrase system.

Streptomyces phage phiC31 integrase is widely used to mediate the integration of exogenous genes into host genomes for gene therapy and genomic modification, as it autonomously performs efficient, unidirectional, site-specific integration into pseudo attP sites of the host genome. Although pseudo attP sites are rarely found within exons, it is necessary to map their precise locations to avoid the risk of insertion mutagenesis. High-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) is a technique that has been developed to recover genomic sequences that flank insertion tags. We have found, however, that this technique is poorly efficient, as it amplifies many non-specific targets and frequently does not generate sufficient product for downstream analysis. Therefore, we have modified the hiTAIL-PCR procedure and re-designed the random primers. As a result, both the amount and specificity of the reaction product were enhanced for each integration site. Restriction analysis of known sequences within the integrated vector, which co-amplified with the flanking genomic sequences, validated 90% of these bands for sequencing. In contrast, only 30% of the bands produced by previous hiTAIL-PCR could be validated. Compared with the original hiTAIL-PCR, our improved hiTAIL-PCR procedure identified phiC31 integration sites more accurately and efficiently.

Reconstructed embryos derived from intersubspecies somatic cell nuclear transfer (SCNT) have poorer developmental potential than those from intrasubspecies SCNT. Based on our previous study that Holstein dairy bovine (HD) mitochondrial DNA (mtDNA) haplotype compatibility between donor karyoplast and recipient cytoplast is crucial for SCNT embryo development, we performed intersubspecies SCNT using HD as donor karyoplast and Luxi yellow heifer (LY) as recipient cytoplast according to mtDNA haplotypes determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The results demonstrated that intersubspecies mtDNA homotype SCNT embryos had higher pre- and post-implantation developmental competence than intrasubspecies mtDNA heterotype embryos as well as improved blastocyst reprogramming status, including normal H3K9 dimethylation pattern and promoter hypomethylation of pluripotent genes such as Oct4 and Sox2, suggesting that intersubspecies SCNT using LY oocytes maintains HD cloning efficiency and may reprogram HD nuclei to develop into a normal cloned animal ultimately. Our results indicated that karyoplast-cytoplast interactions and mtDNA haplotype compatibility may affect bovine intersubspecies SCNT efficiency. This study on bovine intersubspecies SCNT is valuable for understanding the mechanisms of mtDNA haplotype compatibility between karyoplast and cytoplast impacting the bovine SCNT efficiency, and provides an alternative and economic resource for HD cloning.