Efficiency Of Transgenesis Research Articles

Egg Cooling After Oviposition Extends the Permissive Period for Microinjection-Mediated Genome Modification in Bombyx mori

In general, transgenesis efficiency is largely dependent on the developmental status of eggs for microinjection. We investigated whether the relationship between transgenesis efficiency and cooling eggs in silkworms, Bombyx mori, affects the transgenesis frequencies. First, we performed a microinjection using eggs of different developmental statuses at 25 °C. As a result, the use of eggs at 4 h after egg-laying (hAEL) demonstrated nearly five times greater efficiency in frequency compared to 8 hAEL but no transgenesis was found at 12 hAEL. Second, we examined the use of eggs stored for 5 or 24 h at 10 °C. We found that transgenic silkworms were produced not only 5 hAEL but also 24 hAEL. Finally, in the BmBLOS2 gene knock-out experiment, eggs stored at 10 °C demonstrated knock-out phenotypes even 48 hAEL at the time of injection (G0). These results demonstrate that an egg cooling treatment enables drastically enhanced rates of efficiency for insect genome modification. Our results could be useful in other insects, especially species with an extremely short syncytial preblastodermal stage.

Serial Recombineering Cloning to Build Selectable and Tagged Genomic P[acman] BAC Clones for Selection Transgenesis and Functional Gene Analysis using Drosophila melanogaster.

Transgenes with genomic DNA fragments that encompass genes of interest are the gold standard for complementing null alleles in rescue experiments in the fruit fly Drosophila melanogaster. Of particular interest are genomic DNA clones available as bacterial artificial chromosomes (BACs) or fosmids from publicly available genomic DNA libraries. Genes contained within BAC and fosmid clones can be easily modified by recombineering cloning to insert peptide or protein tags to localize, visualize, or manipulate gene products, and to create point mutations or deletions for structure-function analysis of the inserted genes. However, since transgenesis efficiency is inversely correlated with transgene size, obtaining transgenic animals for increasingly larger BAC and fosmid clones requires increasingly laborious screening efforts using the transgenesis marker commonly used for these transgenes, the dominant eye color marker white+ . We recently described a drug-based selectable genetic platform for Drosophila melanogaster, which included four resistance markers that allow direct selection of transgenic animals, eliminating the need to identify transgenic progeny by laborious phenotypic screening. By integrating these resistance markers into BAC transgenes, we were able to isolate animals containing large transgenes by direct selection, avoiding laborious screening. Here we present procedures on how to upgrade BAC clones by serial recombineering cloning to build both selectable and tagged BAC transgenes, for selection transgenesis and functional gene analysis, respectively. We illustrate these procedures using a BAC clone encompassing the gene encoding the synaptic vesicle protein, cysteine string protein. We demonstrate that the modified BAC clone, serially recombineered with a selectable marker for selection transgenesis and an N-terminal green fluorescent protein tag for gene expression analysis, is functional by showing the expression pattern obtained after successful selection transgenesis. The protocols cover: (1) cloning and preparation of the recombineering templates needed for serial recombineering cloning to incorporate selectable markers and protein tags; (2) preparing electrocompetent cells needed to perform serial recombineering cloning; and (3) the serial recombineering workflow to generate both selectable and tagged genomic BAC reporter transgenes for selection transgenesis and functional gene analysis in Drosophila melanogaster. The protocols we describe can be easily adapted to incorporate any of four selectable markers, protein tags, or any other modification for structure-function analysis of the genes present within any of the BAC or fosmid clones. A protocol for generating transgenic animals using serially recombineered BAC clones is presented in an accompanying Current Protocols article (Venken, Matinyan, Gonzalez, & Dierick, 2023a). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning and preparation of recombineering templates used for serial recombineering cloning. Basic Protocol 2: Making electrocompetent cells of the bacterial strains used to perform serial recombineering cloning or induction of plasmid copy number. Basic Protocol 3: Serial recombineering cloning to generate both selectable and tagged genomic P[acman] BAC reporter transgenes for selection transgenesis and gene expression analysis in Drosophila melanogaster.

Highly efficient transgenesis with miniMos in Caenorhabditis briggsae.

Caenorhabditis briggsae as a companion species for Caenorhabditis elegans has played an increasingly important role in study of evolution of development and genome and gene regulation. Aided by the isolation of its sister spices, it has recently been established as a model for speciation study. To take full advantage of the species for comparative study, an effective transgenesis method especially those with single-copy insertion is important for functional comparison. Here, we improved a transposon-based transgenesis methodology that had been originally developed in C. elegans but worked marginally in C. briggsae. By incorporation of a heat shock step, the transgenesis efficiency in C. briggsae with a single-copy insertion is comparable to that in C. elegans. We used the method to generate 54 independent insertions mostly consisting of a mCherry tag over the C. briggsae genome. We demonstrated the use of the tags in identifying interacting loci responsible for hybrid male sterility between C. briggsae and Caenorhabditis nigoni when combined with the GFP tags we generated previously. Finally, we demonstrated that C. briggsae tolerates the C. elegans toxin, PEEL-1, but not SUP-35, making the latter a potential negative selection marker against extrachromosomal array.

An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination.

Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.

Optimized CRISPR tools and site-directed transgenesis towards gene drive development in Culex quinquefasciatus mosquitoes

Culex mosquitoes are a global vector for multiple human and animal diseases, including West Nile virus, lymphatic filariasis, and avian malaria, posing a constant threat to public health, livestock, companion animals, and endangered birds. While rising insecticide resistance has threatened the control of Culex mosquitoes, advances in CRISPR genome-editing tools have fostered the development of alternative genetic strategies such as gene drive systems to fight disease vectors. However, though gene-drive technology has quickly progressed in other mosquitoes, advances have been lacking in Culex. Here, we develop a Culex-specific Cas9/gRNA expression toolkit and use site-directed homology-based transgenesis to generate and validate a Culex quinquefasciatus Cas9-expressing line. We show that gRNA scaffold variants improve transgenesis efficiency in both Culex quinquefasciatus and Drosophila melanogaster and boost gene-drive performance in the fruit fly. These findings support future technology development to control Culex mosquitoes and provide valuable insight for improving these tools in other species.

Improving Transgenesis Efficiency and CRISPR-Associated Tools Through Codon Optimization and Native Intron Addition in Pristionchus Nematodes.

A lack of appropriate molecular tools is one obstacle that prevents in-depth mechanistic studies in many organisms. Transgenesis, clustered regularly interspaced short palindromic repeats (CRISPR)-associated engineering, and related tools are fundamental in the modern life sciences, but their applications are still limited to a few model organisms. In the phylum Nematoda, transgenesis can only be performed in a handful of species other than Caenorhabditis elegans, and additionally, other species suffer from significantly lower transgenesis efficiencies. We hypothesized that this may in part be due to incompatibilities of transgenes in the recipient organisms. Therefore, we investigated the genomic features of 10 nematode species from three of the major clades representing all different lifestyles. We found that these species show drastically different codon usage bias and intron composition. With these findings, we used the species Pristionchus pacificus as a proof of concept for codon optimization and native intron addition. Indeed, we were able to significantly improve transgenesis efficiency, a principle that may be usable in other nematode species. In addition, with the improved transgenes, we developed a fluorescent co-injection marker in P. pacificus for the detection of CRISPR-edited individuals, which helps considerably to reduce associated time and costs.

Live-imaging Analyses Using Small Fish Models Reveal New Mechanisms That Regulate Primary Tumorigenesis

Since the 1980s, zebrafish (Danio rerio) have been used as a valuable model system to investigate developmental processes because they: 1) grow outside their mothers; 2) are transparent during the embryonic stage; and 3) have organs similar to those in humans. Recently, zebrafish have emerged as a powerful model animal for studying not only developmental biology but also human diseases, especially cancer. Owing to the significant advantages of zebrafish, such as low-cost breeding, high efficiency of transgenesis, and ease of in vivo imaging and oncogenic/tumor cell induction, zebrafish offer a unique opportunity to unveil novel mechanisms of cancer progression, invasion, and metastasis. In addition, the small size of zebrafish larvae enables high-throughput chemical screening, and this advantage contributes to generating useful platforms for antitumor drug discovery. Owing to these various merits, which other model animals (such as fly, mouse, and rat) do not possess, zebrafish could achieve a unique status in cancer research. In this review, we discuss the availability of zebrafish for studying cancer and introduce recent cancer studies that have used zebrafish.

PiggyBac Transposon-Mediated Transgenesis in the Pacific Oyster (Crassostrea gigas) - First Time in Mollusks.

As an effective method of transgenesis, the plasmid of PiggyBac transposon containing GFP (PiggyBac) transposon system has been widely used in various organisms but not yet in mollusks. In this work, piggyBac containing green fluorescent protein (GFP) was transferred into the Pacific oyster (Crassostrea gigas) by sperm-mediated gene transfer with or without electroporation. Fluorescent larvae were then observed and isolated under an inverted fluorescence microscope, and insertion of piggyBac was tested by polymerase chain reaction (PCR) using genomic DNA as template. Oyster larvae with green fluorescence were observed after transgenesis with or without electroporation, but electroporation increased the efficiency of sperm-mediated transgenesis. Subsequently, the recombinant piggyBac plasmid containing gGH (piggyBac-gGH) containing GFP and a growth hormone gene from orange-spotted grouper (gGH) was transferred into oysters using sperm mediation with electroporation, and fluorescent larvae were observed and isolated. The insertion of piggyBac-gGH was tested by PCR and genome walking analysis. PCR analysis indicated that piggyBac-gGH was transferred into oyster larvae; genome walking analysis further showed the detailed location where piggyBac-gGH was inserted in the oyster genome. This is the first time that piggyBac transposon-mediated transgenesis has been applied in mollusks.

RNAi-Mediated Loss of Function of Xenopus Immune Genes by Transgenesis.

Generation of transgenic frogs through the stable integration of foreign DNA into the genome is well established in Xenopus This protocol describes the combination of transgenesis with stable RNA interference as an efficient reverse genetic approach to study gene function in Xenopus Initially developed in the fish medaka and later adapted to Xenopus, this transgenic method uses the I-SceI meganuclease, a "rare-cutter" endonuclease with an 18 bp recognition sequence. In this protocol, transgenic X. laevis with knocked down expression of a specific gene are generated using a double promoter expression cassette. This cassette, which is flanked by I-SceI recognition sites, contains the shRNA of choice under the control of the human U6 promoter and a green fluorescent protein (GFP) reporter gene under the control of the human EF-1α promoter. Prior to microinjection the plasmid is linearized by digestion with I-SceI and the entire reaction is then microinjected into one-cell stage eggs. The highly stringent recognition sequence of I-SceI is thought to maintain the linearized plasmid in a nonconcatamerized state, which promotes random integration of the plasmid transgene in the genome. The injected embryos are reared until larval stage 56 and then screened for GFP expression by fluorescence microscopy and assessed for effective knockdown by quantitative RT-PCR using a tail biopsy. Typically, the I-SceI meganuclease transgenesis technique results in 35%-50% transgenesis efficiency, a high survival rate (>35%) and bright nonmosaic GFP expression. A key advantage of this technique is that the high efficiency and nonmosaic transgene expression permit the direct use of F0 animals.

200 Optimization of Individual Stages of Chicken Transgenesis to Increase Efficiency

Primordial germ cells (PGC) are the precursors of male and female progenitor cells. The cells are considered a valuable genetic material for the production of transgenic poultry. This technology includes isolation of the PGC from chick donor embryos, transformation of the cells, and injection into the dorsal aorta of recipient embryos. After injection, the PGC are involved in the process of embryo development and differentiate into male or female sex cells. The aim of the research was to optimize the individual stages of this technology to increase the efficiency of transgenesis. The PGC were extracted from embryo gonads at stage 26 to 27 (H&H) using the trypsinization process. The trypsin concentration and incubation time were determined experimentally. Treatment of chick embryos with a 0.05% trypsin solution for 5 min was optimal for obtaining culture of embryonic cells. Separation of the PGC from other types of embryonic cells was based on a differential adhesive capacity. The maximum homogeneity of the cell population for further cultivation was established by transfer (twice) of the supernatant containing unattached cells after 1 h of cultivation in a new culture dish. The cell population is represented mainly by the PGC (81 ± 4%). Additional purification of the PGC from other cell types using magnetic-activated cell sorting (MACS) increased the proportion of these cells up to 93 ± 2%. The lentiviral transduction (pHAGE vector, ZsGreen under CMV promotor) was used to transform the resulting culture of the PGC. The efficiency of infection of PGC with lentiviral particles (TU/mL = 2.5 × 108) was 70 ± 3%. The transformed cells were injected into the dorsal aorta of recipient embryos on Day 2.5 (n = 80). Before injecting donor PGC, recipient embryos were treated with busulfan to remove the endogenous PGC. The optimal dose of busulfan was selected experimentally. A series of experiments introducing busulfan in concentrations from 50 to 250 μg into chick embryos at 24 h of incubation showed that the optimal dose was 100 μg/embryo. The efficacy of colonization of gonads with donor PGC was assessed on Day-10 embryos (n = 32) and 4-week-old hatched chickens (n = 12). Cells from gonads were studied using fluorescence microscopy, fluorescence-activated cell sorting (FACS) and qPCR. The presence of fluorescent cells in the gonads of recipients was established in both embryos and hatched chickens. The relative number of the recombinant DNA copies and the relative level of expression were confirmed by qPCR. The FACS analysis of sex cells isolated from gonads of recipients showed that the percentage of transformed germ cells reached 55.8% in females (n = 5) and 31.9% in males (n = 7). Thus, the effectiveness of poultry transgenesis can be enhanced by preparation of donor PGC for injection into embryo recipients and elimination of endogenous PGC in recipients. Both the purification of PGC from other cell types based on adhesive capacity as well as treatment of embryo recipients at 24 h incubation with busulfan (100 μg/embryo) increased the effectiveness of transgenesis. Study supported by the RSF within project No. 16-16-10059.

New Wistar Kyoto and Spontaneously Hypertensive rat transgenic models with ubiquitous expression of green fluorescent protein

ABSTRACTThe Wistar Kyoto (WKY) rat and the spontaneously hypertensive (SHR) rat inbred strains are well-established models for human crescentic glomerulonephritis (CRGN) and metabolic syndrome, respectively. Novel transgenic (Tg) strains add research opportunities and increase scientific value to well-established rat models. We have created two novel Tg strains using Sleeping Beauty transposon germline transgenesis, ubiquitously expressing green fluorescent protein (GFP) under the rat elongation factor 1 alpha (EF1a) promoter on the WKY and SHR genetic backgrounds. The Sleeping Beauty system functioned with high transgenesis efficiency; 75% of new rats born after embryo microinjections were transgene positive. By ligation-mediated PCR, we located the genome integration sites, confirming no exonic disruption and defining a single or low copy number of the transgenes in the new WKY-GFP and SHR-GFP Tg lines. We report GFP-bright expression in embryos, tissues and organs in both lines and show preliminary in vitro and in vivo imaging data that demonstrate the utility of the new GFP-expressing lines for adoptive transfer, transplantation and fate mapping studies of CRGN, metabolic syndrome and other traits for which these strains have been extensively studied over the past four decades.

EFFICIENCY OF LOCAL TRANSGENESIS OF THE OVIDUCTAL CELLS IN CHICKEN AS INFLUENCED BY HORMONAL STIMULATION

A b s t r a c t One of the promising areas of biotechnology is to create transgenic chicken bioreactors. However, despite notable successes in avian transgenesis, the creation of transgenic chickens now is a particular problem. Searches and development of alternative methods of directed gene transfer are required, one of which is genetic transformation of certain organs, in particular chicken oviduct (somatic transgenesis). This can significantly reduce the expenditure of time and materials in the preparation of transgenic organisms than using other cellular targets for targeted delivery of DNA (cell blastoderm, primordial germ cells, embryonic stem cells) because the genetically engineering manipulation is possible to be conducted only on embryonic material. The aim of this study was to investigate the effectiveness of the delivery of recombinant DNA into the chicken oviduct cells in vivo and development of methodological approaches to increase the efficiency of transgenesis. In the present work a retroviral vector pLN-GFP was used, based on the Moloney murine leukemia virus (Mo-MuLV), in which a sequence of GFP (green fluorescent protein) marker gene has been cloned. Packaging cell line GP+envAM12 has been used to package retroviral vector pLN-GFP. To determine the optimal duration of administration of gene constructs a proliferative activity was analyzed in the oviduct cells at the age of 1, 2, 3, 4, 4.5 and 5 months, and at the age of 2 months within 24 and 48 hours after hormonal stimulation by 0.1 % sinestrol solution. The introduction of the retroviral vector was performed by injecting the solution of the gene construct directly into the protein part of the oviduct of hens at the age of 4 months without hormonal pre-stimulation (group I) and at the age of 2 months within 24 h after sinestrol injection (group II). Analysis of the integration and expression of recombinant DNA in chick oviduct cells was performed at the age of 6 months. As a result of histological investigations it was found that the maximum proliferative activity in the protein part of oviduct (magnum) was observed in a period between 4 and 4.5 months: the relative DNA content of the cells at this period increased by 3.4 rel. units, which was equivalent to an increase of this parameter over the entire preceding period from 1 to 4 months. The analysis of histological sections of the oviduct in 2-month-old chickens 24 hours after sinestrol injection revealed a significant change in the structure of the oviduct, typical for histological structure of the magnum portion of the oviduct at the 4 month age, and after 48 hours the oviduct histological structure corresponded to that of mature chicken. An average efficiency of the hen oviduct cell transformation assessed as a percentage of the transformed cells to the total cell number of this type in the oviduct, in group I was 17.2±3.1 % whereas in group II it was 57.3±6.3 %. Thus, the use of hormone treatment has allowed a 3.3-fold increase in the effectiveness of local transgenesis of the oviductal cells in chickens.

Isolation and characterization of trophoblast-derived stem-like cells from peri-implantation porcine embryos

In mammals, the trophoblast lineage of the embryo is specified before attachment/implantation to become the fetal portion of the placenta. Trophoblast-derived cells were isolated and cultured from day 10 and day 13 porcine embryos and were grown in vitro in a defined, serum-free culture medium for over 2 years without showing any signs of senescence. However, trophoblast-derived cells placed into serum-containing medium rapidly senesce and fail to proliferate. Semiquantitative and quantitative gene expression analyses of cells in culture from 0 to 30 days confirmed the presence (and relative abundance) of mRNA transcripts from genes involved in trophoblast function (CDX2, TEAD4, CYP17A1, HSD17B1, FGFR2, PLET, HAND1) as well as some genes known to mediate pluripotency (POU5F1, KLF4, CMYC). Protein immunolocalization demonstrated expression of both trophoblast and mesenchymal cell markers. DNA methylation patterns in promoters of three critical developmental genes (HAND1, KLF4, TEAD4) did not change appreciably over 4 months of culture in vitro. It was demonstrated that these trophoblast-derived cells are easily stably transfected with an exogenous transgene (eGFP) by a variety of methods, and show the ability to survive and to be passaged repeatedly after transfection. In summary, early embryonic porcine trophoblast-derived cells have demonstrated unique characteristics, which means they could be used as valuable tools for laboratory work. Anticipated applications include the study of trophoblast physiology as well as possible solutions for improving efficiency of transgenesis by somatic cell nuclear transfer and for pluripotency reprogramming of cells.

A piggyBac transposon‐ and gateway‐enhanced system for efficient BAC transgenesis

Background: Bacterial artificial chromosomes (BACs) have become increasingly popular vectors for making transgenic mice, as they are able to carry large genomic DNA fragments that in many cases are needed to reproduce the endogenous gene expression pattern. However, the efficiency of BAC transgenesis is generally low, and gene transfer to BAC vectors by recombination‐mediated engineering (recombineering) is time‐consuming and technically demanding. Results and Conclusions: We present an enhanced system, comprising a BAC vector retrofitted with piggyBac DNA transposon elements and attL (Gateway) docking sites, that obviates these problems. Using this system, a gene‐of‐interest (such as a reporter gene) is transferred to the vector in a one‐step in vitro reaction, and piggyBac transposition mediates transgene integration at high efficiency when microinjected into mouse zygotes with piggyBac transposase mRNA. We establish proof‐of‐principle for this system using a Wilms tumour‐1 (Wt1) BAC to drive expression of an mCherry‐2A‐EGFP (RG) reporter gene, which yielded transgenic mice at a frequency of 33%, and recapitulated endogenous WT1 expression in developing gonads, kidneys and heart. The system we describe is applicable to any BAC transgenesis strategy. Developmental Dynamics 243:1086–1094, 2014. © 2014 Wiley Periodicals, Inc.

A piggyBac transposon- and gateway-enhanced system for efficient BAC transgenesis.

Bacterial artificial chromosomes (BACs) have become increasingly popular vectors for making transgenic mice, as they are able to carry large genomic DNA fragments that in many cases are needed to reproduce the endogenous gene expression pattern. However, the efficiency of BAC transgenesis is generally low, and gene transfer to BAC vectors by recombination-mediated engineering (recombineering) is time-consuming and technically demanding. We present an enhanced system, comprising a BAC vector retrofitted with piggyBac DNA transposon elements and attL (Gateway) docking sites, that obviates these problems. Using this system, a gene-of-interest (such as a reporter gene) is transferred to the vector in a one-step in vitro reaction, and piggyBac transposition mediates transgene integration at high efficiency when microinjected into mouse zygotes with piggyBac transposase mRNA. We establish proof-of-principle for this system using a Wilms tumour-1 (Wt1) BAC to drive expression of an mCherry-2A-EGFP (RG) reporter gene, which yielded transgenic mice at a frequency of 33%, and recapitulated endogenous WT1 expression in developing gonads, kidneys and heart. The system we describe is applicable to any BAC transgenesis strategy.

English

Lentiviral vectors are an effective method of introducing transgenes into the genome of early stage embryos because they transduce both dividing and non-dividing cells. Lentiviral pseudoparticles containing the coding sequence for the fluorescent protein DsRed were injected into freshly laid leopard gecko eggs. Tissue samples were collected from hatchlings, and the samples were tested for the presence of the transgene. Of the injected gecko population, greater than 89% of efficiency of transgenesis was confirmed using polymerase chain reaction (PCR). Histological evaluations revealed the presence of DsRed 2 in injected gecko organs; with protein production concentrated in the muscle, kidney, and heart. Therefore, lentiviral vectors appear to be viable technology to create transgenic geckos. Key words: DsRed, Eublepharis macularius, Feline Immunodeficienty Virus (FIV), lentiviral transgenesis, reptiles.

The study of factors affecting the efficiency of somatic transgenesis in chickens

In this article we studied the impact of the use of retroviral vectors for genetic transformation of chicken oviduct cells in vivo to produce transgenic chickens bioreactors and investigated factors affecting the efficiency of local transgenesis in chickens. By using of retroviral vector system it was shown that with the efficiency of chickens oviduct cells transformation was at least 17,2 ± 3,1%.

Optimization of transgenesis conditions for the generation of CXCL2-luciferase reporter mice line

Background: Transgenesis by microinjection has been widely used for the generation of different mouse models. Different variables of the procedure may critically affect the efficiency of the process. A DNA construction that carries the CXCL2 promoter gene and firefly luciferase has been used to optimize aspects of the procedure. Three different concentrations (0.5, 1.0 and 4.0 ng/µl) of the DNA construction to microinject a total of 1981 zygotes has been tested. Intact/injected embryos, pregnancy and birth rate, survival of pups 7 days after birth, number of transgenic pups and overall transgenic efficiency was registered and analyzed by Z test of proportions for each group. Results: A total of seven transgenic founders were detected for the three DNA concentrations used, 1 in 46 alive pups in the 0.5 ng/ml group, 5 in 38 alive pups in the 1 ng/ml group and 1 in 21 alive pups in the 4 ng/ml group ( p Conclusions: In conclusion, we have selected the best operative conditions to maximize the transgenesis efficiency. Furthermore, the transgenic lines developed could be used as a reporter model of innate immunity activation with many different applications in the fields of immunology, cancer and neurodegenerative diseases.

Advanced transgenic strategies for modification of donor pigs in xenotransplantation

For xenotransplantation diverse rejection mechanisms are much more pronounced as compared to allotransplantation. The usage of genetically modified pigs, however, facilitates tailoring of donor animals for defined purposes (1). Although such genetic modifications were done decades ago, routine generation of transgenic pigs was not performed until somatic cell nuclear transfer (SCNT) was implemented for reproduction of large animals. SCNT avoids the production of mosaic founders and shifted the genetic modification towards the level of pig primary cells and, thus, improved the efficiency of transgenesis as it enables the generation of almost exclusively transgenic offspring, once the donor cells have been properly selected for vector integration (2).In addition, SCNT opened up the possibilities for any type of genetic modifications that has been developed for embryonic stem cells. In particular, the opportunity for site‐directed mutagenesis boosted the potential of genetically modified pig models. This was demonstrated for the removal of the α1,3‐galactosyl‐galactose epitopes by disruption of the GGTA1 gene which reduced the problem of hyperacute rejection to a minor topic in the xenotransplantation community. In the meanwhile more sophisticated methods such as modified bacterial artificial chromosomes, viral vectors or site‐specific nucleases further increased the potential for site‐directed mutagenesis in pig (3, 4). The latter technology is based on the introduction of a DNA double strand break by a nuclease that is directed to the target site by specific DNA‐binding domains. Mutations are introduced by erroneous repair through non‐homologous end joining. Alternatively, a targeting vector can be used in combination with a site‐specific nuclease to introduce a targeted modification via homologous recombination. Other advanced transgenic strategies such as the two‐vector based TetOn technology for inducible transgene expression are routinely performed in the mouse, but the significantly longer generation time of large animals hampered its straight translation into the pig. As we demonstrated recently, sequential transgenesis by repeated rounds of SCNT is a practicable way to evaluate biological transgene function in founder animals within a considerable time frame (5).In addition to technological improvements at the cellular as well as at the embryonic level, the recent boost of genomic information from multiple species and its bioinformatics analysis improved the design of transgenic pigs. As for many problems cell type‐specific expression of a transgene is desired, the definition of appropriate regulatory elements is required. Many of those have been described in the mouse, but in general endogenous sequences are seen as superior to the usage of murine promoters in the pig. Multiple‐sequence alignments from diverse mammalian species facilitate the identification of the orthologous region of murine regulatory elements in the pig.Interestingly, with the increasing number of transgenes available for xenotransplantation approaches, the breeding aspect gained new attention. It is clear that for optimized donor pigs multiple transgenes should be combined and, on the long run, mendelian transgene segregation should be avoided by using novel transgene approaches. However, until such “all‐in‐one” vectors are available, the most straightforward strategy is the combination of existing and properly characterized lines by conventional breeding strategies. These require profound organization and logistics to resolve the conflicting aspects of transgene segregation and inbreeding and to enable the systematic evaluation of donor herds for microbial contamination.Thus, the task field of donor pig suppliers in xenotransplantation expanded from relatively simple reproductive stints to advanced design and construction of novel transgenic pigs and organizing challenges regarding continuous supply of donor animals.

ОПТИМИЗАЦИЯ УСЛОВИЙ ВВЕДЕНИЯ РЕКОМБИНАНТНОЙ ДНК В СПЕРМАТОГЕННЫЕ КЛЕТКИ СЕМЕННИКОВ ПЕТУХОВ in vivo

The factors affecting the efficiency of the transfer of recombinant DNA into seminiferous epithelium of the chicken in vivo using retroviral vectors are studied. The maximum efficiency of the transformation of spermatogenic cells in chicken is demonstrated when virus was used as a source of genetic structure (pLgfpSN gene construct which contains the reporter gene of green fluorescent protein GFP based on the recombinant retroviral vector). The possibility of transmission of the transgene to offspring with the efficiency of transgenesis 4.2 % is shown.