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Digital agriculture (DA) can contribute solutions to meet an increase in healthy, nutritious, and affordable food demands in an efficient and sustainable way. South America (SA) is one of the main grain and protein producers in the world but the status of DA in the region is unknown. A systematic review and case studies from Brazil, Argentina, Uruguay, and Chile were conducted to address the following objectives: (1) quantify adoption of existing DA technologies, (2) identify limitations for DA adoption; and (3) summarise existing metrics to benchmark DA benefits. Level of DA adoption was led by Brazil and Argentina followed by Uruguay and at a slower rate, Chile. GPS guidance systems, mapping tools, mobile apps and remote sensing were the most adopted DA technologies in SA. The most reported limitations to adoption were technology cost, lack of training, limited number of companies providing services, and unclear benefits from DA. Across the case studies, there was no clear definition of DA. To mitigate some of these limitations, our findings suggest the need for a DA educational curriculum that can fulfill the demand for job skills such as data processing, analysis and interpretation. Regional efforts are needed to standardise these metrics. This will allow stakeholders to design targeted initiatives to promote DA towards sustainability of food production in the region. The status of digital agriculture (DA) in South America (SA) is unknown. This article quantified adoption of existing DA technologies, identified limitations for DA adoption, implementing a literature review and case studies. Sustainable metrics were summarised to benchmark DA impact. GPS guidance systems, mapping tools, mobile apps and remote sensing were the most adopted DA technologies in SA. The most reported limitations to adoption were technology cost, lack of training, limited number of companies providing services, and unclear benefits from DA. The proposed metrics to benchmark DA benefits will allow stakeholders to design targeted initiatives to promote DA towards sustainability of food production in the region. [ABSTRACT FROM AUTHOR]

Achieving regeneration in humans has been a long-standing goal of many researchers. Whereas amphibians like the axolotl ( Ambystoma mexicanum ) are capable of regenerating whole organs and even limbs, most mammals heal their wounds via fibrotic scarring. Recently, the African spiny mouse ( Acomys sp.) has been shown to be injury resistant and capable of regenerating several tissue types. A major focal point of research with Acomys has been the identification of drivers of regeneration. In this search, the matrisome components related to the extracellular matrix (ECM) are often overlooked. In this review, we compare Acomys and axolotl skin wound healing and blastema-mediated regeneration by examining their wound healing responses and comparing the expression pattern of matrisome genes, including glycosaminoglycan (GAG) related genes. The goal of this review is to identify matrisome genes that are upregulated during regeneration and could be potential candidates for inclusion in pro-regenerative biomaterials. Research papers describing transcriptomic or proteomic coverage of either skin regeneration or blastema formation in Acomys and axolotl were selected. Matrisome and GAG related genes were extracted from each dataset and the resulting lists of genes were compared. In our analysis, we found several genes that were consistently upregulated, suggesting possible involvement in regenerative processes. Most of the components have been implicated in regulation of cell behavior, extracellular matrix remodeling and wound healing. Incorporation of such pro-regenerative factors into biomaterials may help to shift pro-fibrotic processes to regenerative responses in treated wounds.

The African spiny mouse (Acomys cahirinus) is an emerging model of mammalian epimorphic regeneration that has aroused the interest of the scientific community in the last decade. To date, studies on brain repair have been hindered by the lack of knowledge on the neuroanatomy of this species. Here, we present a coronal brain atlas in stereotaxic coordinates, which allows for three-dimensional identification and localization of the brain structures of this species. The brain of 12-week-old spiny mice was mapped in stereotaxic coordinates using cresyl violet-stained brain sections obtained from coronal cryosectioning of the brain after transcardial perfusion with fixative. The atlas is presented in 42 plates representing sections spaced 240 μm apart. Stereotaxic coordinates were validated using both a model of Parkinsonian lesion of the striatum with 6-hydroxydopamine and labeling of the corticospinal tract in the spiny mouse spinal cord using AAV1/2-GFP intracortical injections. This work presents a new tool in A. cahirinus neurobiology and opens new avenues of research for the investigation of the regenerative ability of A. cahirinus in models of brain disorders., (© 2022 Wiley Periodicals LLC.)

For the estimation of the soil organic carbon stocks, bulk density (BD) is a fundamental parameter but measured data are usually not available especially when dealing with legacy soil data. It is possible to estimate BD by applying pedotransfer function (PTF). We applied different estimation methods with the aim to define a suitable PTF for BD of arable land for the Mediterranean Basin, which has peculiar climate features that may influence the soil carbon sequestration. To improve the existing BD estimation methods, we used a set of public climatic and topographic data along with the soil texture and organic carbon data. The present work consisted of the following steps: i) development of three PTFs models separately for top (0-0.4 m) and subsoil (0.4-1.2 m), ii) a 10-fold cross-validation, iii) model transferability using an external dataset derived from published data. The development of the new PTFs was based on the training dataset consisting of World Soil Information Service (WoSIS) soil profile data, climatic data from WorldClim at 1 km spatial resolution and Shuttle Radar Topography Mission (SRTM) digital elevation model at 30 m spatial resolution. The three PTFs models were developed using: Multiple Linear Regression stepwise (MLR-S), Multiple Linear Regression backward stepwise (MLR-BS), and Artificial Neural Network (ANN). The predictions of the newly developed PTFs were compared with the BD calculated using the PTF proposed by Manrique and Jones (MJ) and the modelled BD derived from the global SoilGrids dataset. For the topsoil training dataset (N = 129), MLR-S, MLR-BS and ANN had a R 2 0.35, 0.58 and 0.86, respectively. For the model transferability, the three PTFs applied to the external topsoil dataset (N = 59), achieved R 2 values of 0.06, 0.03 and 0.41. For the subsoil training dataset (N = 180), MLR-S, MLR-BS and ANN the R 2 values were 0.36, 0.46 and 0.83, respectively. When applied to the external subsoil dataset (N = 29), the R 2 values were 0.05, 0.06 and 0.41. The cross-validation for both top and subsoil dataset, resulted in an intermediate performance compared to calibration and validation with the external dataset. The new ANN PTF outperformed MLR-S, MLR-BS, MJ and SoilGrids approaches for estimating BD. Further improvements may be achieved by additionally considering the time of sampling, agricultural soil management and cultivation practices in predictive models., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Decidualizing endometrial stromal cells (EnSC) critically determine the maternal response to an implanting conceptus, triggering either menstruation-like disposal of low-fitness embryos or creating an environment that promotes further development. However, the mechanism that couples maternal recognition of low-quality embryos to tissue breakdown remains poorly understood. Recently, we demonstrated that successful transition of the cycling endometrium to a pregnancy state requires selective elimination of pro-inflammatory senescent decidual cells by activated uterine natural killer (uNK) cells. Here we report that uNK cells express CD44, the canonical hyaluronan (HA) receptor, and demonstrate that high molecular weight HA (HMWHA) inhibits uNK cell-mediated killing of senescent decidual cells. In contrast, low molecular weight HA (LMWHA) did not attenuate uNK cell activity in co-culture experiments. Killing of senescent decidual cells by uNK cells was also inhibited upon exposure to medium conditioned by IVF embryos that failed to implant, but not successful embryos. Embryo-mediated inhibition of uNK cell activity was reversed by recombinant hyaluronidase 2 (HYAL2), which hydrolyses HMWHA. We further report a correlation between the levels of HYAL2 secretion by human blastocysts, morphological scores, and implantation potential. Taken together, the data suggest a pivotal role for uNK cells in embryo biosensing and endometrial fate decisions at implantation., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Research Question: Which are the early compartment-specific transcriptional responses of the trophoblast and the endometrial epithelium throughout early attachment during implantation?, Design: An endometrial epithelium proxy (cell line Ishikawa) was co-cultured with spheroids of a green fluorescent protein (GFP) expressing trophoblast cell line (JEG-3). After 0, 8 and 24 h of co-culture, the compartments were sorted by fluorescence-activated cell sorting; GFP+ (trophoblast), GFP- (epithelium) and non-co-cultured control populations were analysed (in triplicate) by RNA-seq and gene set enrichment analysis (GSEA)., Results: Trophoblast challenge induced a wave of transcriptional changes in the epithelium that resulted in 295 differentially regulated genes involving epithelial to mesenchymal transition (EMT), cell movement, apoptosis, hypoxia, inflammation, allograft rejection, myogenesis and cell signalling at 8 h. Interestingly, many of the enriched pathways were subsequently de-enriched by 24 h (i.e. EMT, cell movement, allograft rejection, myogenesis and cell signalling). In the trophoblast, the co-culture induced more transcriptional changes and regulation of a variety of pathways. A total of 1247 and 481 genes were differentially expressed after 8 h and from 8 to 24 h, respectively. Angiogenesis and hypoxia were over-represented at both stages, while EMT and cell signalling only were at 8 h; from 8 to 24 h, inflammation and oestrogen response were enriched, while proliferation was under-represented., Conclusions: Successful attachment produced a series of dynamic changes in gene expression, characterized by an overall early and transient transcriptional up-regulation in the receptive epithelium, in contrast to a more dynamic transcriptional response in the trophoblast., (Copyright © 2020 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Research Question: What is the relationship between the vaginal microbiota profile at the time of embryo transfer and live birth rates in women undergoing IVF/intracytoplasmic sperm injection (ICSI) with donated oocytes?, Design: One hundred and fifty Caucasian women receiving donated oocytes were prospectively included in the study from March 2017 to January 2018. Samples of vaginal fluid were taken immediately before transfer of a fresh single blastocyst and genomic DNA (gDNA) was extracted. Bacterial load as well as the presence of four lactobacilli (L. crispatus, L. gasseri, L. jensenii and L. iners) and species associated with bacterial vaginosis (Gardnerella vaginalis, Atopobium vaginae, Mycoplasma hominis and Prevotella spp. - here collectively termed BVB) were determined by quantitative polymerase chain reaction. Vaginal microbiota profiles for each patient were characterized and correlated with reproductive results., Results: Although bacterial load was variable, a majority of samples were dominated by a single species (80.7%, 121/150). Most samples (76.7%, 115/150) were dominated by Lactobacillus spp., while 23.3% (35/150) were dominated by bacteria associated with bacterial vaginosis. The distribution of microbiota profiles among women who achieved a live birth and women who did not was similar (P = 0.43). Interestingly, we found a significantly higher proportion of samples dominated by L. crispatus- in women achieving live birth compared with those who did not (P = 0.021); this correlation was also statistically significant for biochemical pregnancy (P = 0.039) and clinical pregnancy (P = 0.015)., Conclusions: Our data suggest that bacterial vaginosis-like vaginal microbiota at the time of embryo transfer does not directly affect the live birth rate., (Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Purpose: Several in vitro systems have been reported to model human implantation; however, the molecular dynamics of the trophoblast vs. the epithelial substrate during attachment have not been described. We have established an in vitro model which allowed us to dissect the transcriptional responses of the trophoblast and the receptive vs. non-receptive epithelium after co-culture., Methods: We established an in vitro system based on co-culture of (a) immortalized cells representing receptive (Ishikawa) or non-receptive (HEC-1-A) endometrial epithelium with (b) spheroids of a trophoblastic cell line (JEG-3) modified to express green fluorescent protein (GFP). After 48 h of co-culture, GFP+ (trophoblast cells) and GFP- cell fractions (receptive or non-receptive epithelial cells) were isolated by fluorescence-activated flow cytometry (FACS) and subjected to RNA-seq profiling and gene set enrichment analysis (GSEA)., Results: Compared to HEC-1-A, the trophoblast challenge to Ishikawa cells differentially regulated the expression of 495 genes, which mainly involved cell adhesion and extracellular matrix (ECM) molecules. GSEA revealed enrichment of pathways related to cell division, cell cycle regulation, and metabolism in the Ishikawa substrate. Comparing the gene expression profile of trophoblast spheroids revealed that 1877 and 323 genes were upregulated or downregulated when co-cultured on Ishikawa substrates (compared to HEC-1-A), respectively. Pathways favorable to development, including tissue remodeling, organogenesis, and angiogenesis, were enhanced in the trophoblast compartment after co-culture of spheroids with receptive epithelium. By contrast, the co-culture with less receptive epithelium enriched pathways mainly related to trophoblast cell proliferation and cell cycle regulation., Conclusions: Endometrial receptivity requires a transcriptional signature that determines the trophoblast response and drives attachment.

The African spiny mouse ( Acomys spp.) is an emerging animal model with remarkable biological characteristics that make it a subject of interest for a broad range of research fields. Typically a desert species adapted to a low-calorie diet, spiny mice develop diabetes-related symptoms when switched to high-energy diets. Spiny mice undergo relatively long gestation periods and have small litters of highly developed pups, making them an adequate model for late organogenesis and perinatal biology. Recently, they have been shown to have remarkable healing and regeneration capabilities, which make them unique among mammals. In this work, we describe our experience in housing a colony of African spiny mice and cover all basic aspects of feeding, maintenance and breeding for research purposes.

Gaucher's disease (GD) is the most frequently inherited lysosomal storage disease, presenting both visceral and neurologic symptoms. Mutations in acid β-glucocerebrosidase disrupt the sphingolipid catabolic pathway promoting glucosylceramide (GlcCer) accumulation in lysosomes. Current treatment options are enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). However, neither of these approaches is effective in treating the neurological aspect of the disease. The use of small pharmacological compounds that act as molecular chaperones is a promising approach that is still experimental. In recent years, an association between GD and Parkinson like synucleinopathies has been discovered. Since 1992, a number of mouse models of GD have been the developed and partially reproduce phenotype of the disease. More recently, the discovery of direct reprograming has allowed the derivation of induced pluripotent stem cells (iPSc) from fibroblasts obtained from GD patients. iPSc can be expanded indefinitely in vitro and differentiated to macrophages and neurons, the main relevant cell types involved in GD. In this work, we review iPSc models of GD and summarize what we have learned from this system.

Background: Gaucher disease (GD) is caused by mutations in the GBA1 gene which encodes lysosomal β-glucocerebrosidase (GCase). In GD, partial or complete loss of GCase activity causes the accumulation of the glycolipids glucosylceramide (GlcCer) and glucosylsphingosine in the lysosomes of macrophages. In this manuscript, we investigated the effects of glycolipids accumulation on lysosomal and mitochondrial function, inflammasome activation and efferocytosis capacity in a THP-1 macrophage model of Gaucher disease. In addition, the beneficial effects of coenzyme Q 10 (CoQ) supplementation on cellular alterations were evaluated. Chemically-induced Gaucher macrophages were developed by differentiateing THP-1 monocytes to macrophages by treatment with phorbol 12-myristate 13-acetate (PMA) and then inhibiting intracellular GCase with conduritol B-epoxide (CBE), a specific irreversible inhibitor of GCase activity, and supplementing the medium with exogenous GlcCer. This cell model accumulated up to 16-fold more GlcCer compared with control THP-1 cells., Results: Chemically-induced Gaucher macrophages showed impaired autophagy flux associated with mitochondrial dysfunction and increased oxidative stress, inflammasome activation and impaired efferocytosis. All abnormalities were partially restored by supplementation with CoQ., Conclusion: These data suggest that targeting mitochondria function and oxidative stress by CoQ can ameliorate the pathological phenotype of Gaucher cells. Chemically-induced Gaucher macrophages provide cellular models that can be used to investigate disease pathogenesis and explore new therapeutics for GD.

In most species androgens shorten the cardiac action potential and reduce the risk of afterdepolarizations. Despite the central role of the rat model in physiological studies, the effects of androgens on the rat heart are still inconclusive. We therefore performed electrophysiological studies on the perfused rat right ventricular free wall. We found a correlation between androgenic activity and a propensity to generate ventricular ectopic action potentials. We also found that the testosterone treatment increased action potential duration at 90 % of repolarization (APD90), while androgenic inhibition increased the time to peak and decreased APD90. We observed that the voltage-gated potassium channel Kv4.3 and the bi-directional membrane ion transporter NCX in the rat myocardium were regulated by androgenic hormones. One possible explanation for these findings is that due to the expression of specific ion channels in the rat myocardium, the action potential response to its hormonal background is different from those described in other experimental models. Our results indicate that androgenic control of NCX expression plays a key role in determining arrhythmogenicity in the rat heart.

Lysosomal storage diseases (LSDs) describe a heterogeneous group of rare inherited metabolic disorders that result from the absence or loss of function of lysosomal hydrolases or transporters, resulting in the progressive accumulation of undigested material in lysosomes. The accumulation of substances affects the function of lysosomes and other organelles, resulting in secondary alterations such as impairment of autophagy, mitochondrial dysfunction, inflammation and apoptosis. LSDs frequently involve the central nervous system (CNS), where neuronal dysfunction or loss results in progressive neurodegeneration and premature death. Many LSDs exhibit signs of mitochondrial dysfunction, which include mitochondrial morphological changes, decreased mitochondrial membrane potential (ΔΨm), diminished ATP production and increased generation of reactive oxygen species (ROS). Furthermore, reduced autophagic flux may lead to the persistence of dysfunctional mitochondria. Gaucher disease (GD), the LSD with the highest prevalence, is caused by mutations in the GBA1 gene that results in defective and insufficient activity of the enzyme β-glucocerebrosidase (GCase). Decreased catalytic activity and/or instability of GCase leads to accumulation of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph) in the lysosomes of macrophage cells and visceral organs. Mitochondrial dysfunction has been reported to occur in numerous cellular and mouse models of GD. The aim of this manuscript is to review the current knowledge and implications of mitochondrial dysfunction in LSDs.

While regeneration occurs in a number of taxonomic groups across the Metazoa, there are very few reports of regeneration in mammals, which generally respond to wounding with fibrotic scarring rather than regeneration. A recent report described skin shedding, skin regeneration and extensive ear punch closure in two rodent species, Acomys kempi and Acomys percivali. We examined these striking results by testing the capacity for regeneration of a third species, Acomys cahirinus, and found a remarkable capacity to repair full thickness circular punches in the ear pinna. Four-millimeter-diameter wounds closed completely in 2 months in 100% of ear punches tested. Histology showed extensive formation of elastic cartilage, adipose tissue, dermis, epidermis and abundant hair follicles in the repaired region. Furthermore, we demonstrated abundant angiogenesis and unequivocal presence of both muscle and nerve fibers in the reconstituted region; in contrast, similar wounds in C57BL/6 mice simply healed the borders of the cut by fibrotic scarring. Our results confirm the regenerative capabilities of Acomys, and suggest this model merits further attention.

Exposure to air particulate matter (PM) is associated with increased cardiovascular morbimortality. However, PM doesn't affect equally to all people, being the old cohort the most susceptible and studied. We hypothesized that another specific life phase, the middle-aged subpopulation, may be negatively affected. Therefore, the aim of this study was to analyze in vivo the acute biological impact of two environmental particles, Urban Air Particles from Buenos Aires and Residual Oil Fly Ash, on the cardiorespiratory system of middle-aged mice, evaluating oxidative metabolism and inflammation. Both PM provoked a local and systemic inflammatory response, leading to a reduced alveolar area in the lung, an epicard inflammation in the heart, an increment of IL-6, and a reduction on PON 1 activity in serum of middle-aged animals. The positive correlation of local parameters with systemic markers of oxidative stress and inflammation could be responsible for associations of cardiovascular morbimortality in this subpopulation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A general approach is reported for the design of small-molecule competitive inhibitors of lysosomal glycosidases programmed to 1) promote correct folding of mutant enzymes at the endoplasmic reticulum, 2) facilitate trafficking, and 3) undergo dissociation and self-inactivation at the lysosome. The strategy is based on the incorporation of an orthoester segment into iminosugar conjugates to switch the nature of the aglycone moiety from hydrophobic to hydrophilic in the pH 7 to pH 5 window, which has a dramatic effect on the enzyme binding affinity. As a proof of concept, new highly pH-responsive glycomimetics targeting human glucocerebrosidase or α-galactosidase with strong potential as pharmacological chaperones for Gaucher or Fabry disease, respectively, were developed., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The derivation of induced pluripotent cells (iPSCs) from individuals suffering from genetic syndromes offers new opportunities for basic research into these diseases and the development of therapeutic compounds. iPSCs can self renew and can be differentiated to many cell types, offering a potentially unlimited source of material for study. In this review we discuss the conceptual and practical issues to consider when attempting to model genetic diseases using iPSCs.

Since their discovery in the early 1990s, microRNAs (miRs) have gone from initially being considered an oddity to being recognized as a level of gene expression regulation that is integral to the normal function of cells and organisms. They are implicated in many if not all biological processes in animals, from apoptosis and cell signaling to organogenesis and development. Our understanding of cell regulatory states, as determined primarily by transcription factor (TF) profiles, is incomplete without consideration of the corresponding miR profile. The miR complement of a cell provides robust and redundant control over the output of hundreds of possible targets for each miR. miRs are common components of regulatory pathways, and in some cases can constitute on-off switches that regulate crucial fate decisions. In this review, we summarize our current knowledge about the biogenesis and regulation of miRs and describe their involvement in the pathways that regulate cell division, pluripotency, and reprogramming to the pluripotent state.

The generation of induced pluripotent stem (iPS) cells has enabled the derivation of patient-specific pluripotent cells and provided valuable experimental platforms to model human disease. Patient-specific iPS cells are also thought to hold great therapeutic potential, although direct evidence for this is still lacking. Here we show that, on correction of the genetic defect, somatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency to generate patient-specific iPS cells. These cell lines appear indistinguishable from human embryonic stem cells and iPS cells from healthy individuals. Most importantly, we show that corrected Fanconi-anaemia-specific iPS cells can give rise to haematopoietic progenitors of the myeloid and erythroid lineages that are phenotypically normal, that is, disease-free. These data offer proof-of-concept that iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications.

Induced pluripotent stem (iPS) cells have generated keen interest due to their potential use in regenerative medicine. They have been obtained from various cell types of both mice and humans by exogenous delivery of different combinations of Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28. The delivery of these transcription factors has mostly entailed the use of integrating viral vectors (retroviruses or lentiviruses), carrying the risk of both insertional mutagenesis and oncogenesis due to misexpression of these exogenous factors. Therefore, obtaining iPS cells that do not carry integrated transgene sequences is an important prerequisite for their eventual therapeutic use. Here we report the generation of iPS cell lines from mouse embryonic fibroblasts with no evidence of integration of the reprogramming vector in their genome, achieved by nucleofection of a polycistronic construct coexpressing Oct4, Sox2, Klf4, and c-Myc.

The utility of induced pluripotent stem (iPS) cells for investigating the molecular logic of pluripotency and for eventual clinical application is limited by the low efficiency of current methods for reprogramming. Here we show that reprogramming of juvenile human primary keratinocytes by retroviral transduction with OCT4, SOX2, KLF4 and c-MYC is at least 100-fold more efficient and twofold faster compared with reprogramming of human fibroblasts. Keratinocyte-derived iPS (KiPS) cells appear indistinguishable from human embryonic stem cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, global gene expression profiles and differentiation potential in vitro and in vivo. To underscore the efficiency and practicability of this technology, we generated KiPS cells from single adult human hairs. Our findings provide an experimental model for investigating the bases of cellular reprogramming and highlight potential advantages of using keratinocytes to generate patient-specific iPS cells.

INTRODUCTIONThis protocol describes the use of lentiviral vectors to deliver small interfering RNA (siRNA)-mediated silencing cassettes. The combination of these two technologies allows for the development of a powerful tool to achieve long-term down-regulation of specific target genes both in vitro and in vivo. It combines the specificity of RNA interference with the versatility of lentiviral vectors to stably transduce a wide range of cell types. In this method, a small hairpin (shRNA) is cloned initially into an entry vector (pENTR/U6) immediately downstream from an hU6 promoter. The silencing cassette is flanked by recombination sites from bacteriophage λ (attL1 and attL2). Once an effective shRNA is obtained, it can be transferred to the destination vector. The destination vector is a lentiviral vector carrying a marker (green fluorescent protein [GFP] or a selection marker) with a destination cassette cloned upstream of the marker (attR1 and attR2 flanking a ccdB toxic gene). Thus, the silencing cassette can be transferred from the entry vector to the destination vector in a simple Gateway LR cloning reaction. The positioning of the silencing cassette upstream of the marker expression cassette avoids down-regulation of the marker.

INTRODUCTIONThis protocol combines the specificity of small interfering RNA (siRNA)-mediated silencing cassettes with the versatility of lentiviral vectors to stably transduce a wide range of cell types. A short hairpin RNA (shRNA) designed against a given target is cloned into a plasmid containing the pol III promoter. The design uses a 5' forward primer upstream of the pol III promoter and a 3' reverse primer that includes the entire shRNA sequence (i.e., sense, loop, and antisense sequences followed by five Ts), followed by 22 bases complementary to the last 22 bp upstream of the +1 transcriptional start site of the pol III promoter. An NheI-compatible restriction site is included at the 5' end of both forward and reverse primers. A single round of PCR is used to amplify this template. The resulting DNA fragment contains an shRNA expression cassette that can be cloned into a simple cloning vector, tested, and then transferred to the lentiviral vector, or cloned into the lentiviral vector directly. This procedure uses a unique restriction site in the 3' long terminal repeat (LTR). During integration, the 5' LTR of the provirus is copied from the 3' LTR, cloning the H1-driven shRNA into the 3' LTR, resulting in duplication of the silencing cassette. This strategy maximizes the silencing power of the lentiviral vector. The combination of the lentiviral and siRNA technologies provides a powerful tool to achieve long-term down-regulation of specific target genes both in vitro and in vivo.

INTRODUCTIONIn order to generate transgenic and knockout animals, preimplantation embryos must be harvested and manipulated in vitro. Although the production of transgenic animals has been performed by pronuclear injection of DNA into single-cell embryos, the generation of mouse knockouts is time-consuming and laborious. An embryonic stem (ES) knockout line must be generated, characterized, and injected into a blastocyst in order to obtain a chimeric founder that can be subsequently bred to homozygosity. Taking advantage of the unique ability of lentiviral vectors to generate transgenic animals, one can use lentiviruses expressing short hairpin RNAs (shRNAs) from polymerase III (pol III) promoters such as H1 and mU6 to produce transgenic knockdown mice. This protocol describes two methods to deliver genes and small interfering RNA (siRNA)-expressing cassettes into preimplantation mouse embryos using lentiviral vectors: zona pellucida removal and subzonal injection. The zona pellucida removal method is able to achieve up to 100% transgenesis and does not require the use of a micromanipulator. However, zona removal is toxic to the embryos and results in low survival of embryos. The subzonal injection method is able to achieve up to 100% transgenesis and is not toxic for the embryos; therefore, survival of embryos is much higher. Nevertheless, it involves the use of a micromanipulator, which requires some skill.

Lentiviral vectors are potent gene delivery vehicles that enable stable expression of transgenes in both dividing and postmitotic cells, including preimplantation embryos. We have developed lentiviral vectors carrying silencing cassettes consisting of an RNA polymerase III promoter expressing short hairpin RNAs. Transgenic mice can be generated rapidly by transduction of early embryos with lentiviral silencing vectors, resulting in mice with downregulated target genes. We describe two alternative early embryo transduction protocols (removal of zona pellucida and subzonal microinjection). These methodologies offer the possibility of large-scale generation of knockdown transgenic mice for functional genomic studies and enable the production of transgenic mice in 7 weeks.

RNA interference (RNAi) has emerged as a powerful technique to downregulate gene expression. The use of polIII promoters to express small hairpin RNAs (shRNAs), combined with the versatility and robustness of lentiviral vector-mediated gene delivery to a wide range of cell types offers the possibility of long-term downregulation of specific target genes both in vitro and in vivo. The use of silencing lentivectors allows for a rapid and convenient way of establishing cell lines (or transgenic mice) that stably express shRNAs for analysis of phenotypes produced by knockdown of a gene product. Here we present two possible protocols describing the design and cloning of silencing lentiviral vectors. These protocols can be completed in less than 3 weeks.