Retroviral-mediated gene transfer as an effective tool for the in vitro genetic transformation of chicken embryonic cells and production of transgenic chickens

The data on the in vitro and in vivo (into embryonic disk) retroviral-mediated transfer of genetic information into chicken embryonic cells are presented. The estimated transformation frequency of the cultured target cells constituted 8 × 10^?4 to 5 × 10^?3. A transgenic rooster, carrying recombinant DNA in blood, heart, liver, and intestine cells, was obtained.     

Haploid embryonic stem cells: an ideal tool for mammalian genetic analyses

Identification of the function of all genes in the mammalian genome is critical in understanding basic mechanisms of biology. However, the diploidy of mammalian somatic cells has greatly hindered efforts to elucidate the gene function in numerous biological processes by mutagenesis-based genetic approaches. Recently, mouse haploid embryonic stem (haES) cells have been successfully isolated from parthenogenetic and androgenetic embryos, providing an ideal tool for genetic analyses. In these studies, mouse haES cells have already shown that they could be used in cell-based forward or reverse genetic screenings and in generating gene-targeting via homologous recombination. In particular, haES cells from androgenetic embryos can be employed as novel, renewable form of fertilization agent for yielding live-born mice via injection into oocytes, thus showing the possibility that genetic analysis can be extended from cellular level to organism level.     

Cholaminchloride hydrochloride-cationized gelatin/calcium-phosphate nanoparticles as gene carriers for transgenic chicken production

Currently, transgenic chickens are a popular method for pharmaceutical protein production. In this study, we employed cationized gelatin/calcium phosphate nanoparticles (ca-GCaPs) with surface modification by cholaminchloride hydrochloride as gene carriers to produce transgenic chickens. To evaluate the transfection efficiency, a plasmid (pEGFP-C1) was encapsulated in the ca-GCaPs for HeLa cells transfection. The ca-GCaPs were applied as gene carriers by direct injection into the area opaca of the chicken embryo blastodisc to produce the transgenic chicken. The particle size of the ca-GCaPs was 350 nm and the zeta potential was +15 mV. Plasmids encapsulated in the ca-GCaPs prevented from DNase I degradation. Based on biocompatibility analysis, the ca-GCaPs produce higher cell viability and less cytotoxicity compared to the commercially available product Lipofectamine? 2000. The in vitro study indicated that the transfection rate using ca-GCaPs was higher than using cationized gelatin nanoparticles (ca-GPs) because calcium phosphate can provide osmotic pressure to break down lysosomes. In the in vivo study, the egg hatch rate was 100% after ca-GCaPs transfection, and green fluorescence protein expression could be observed in chicken tissue on the fourth day after transfection.     

Retroviral-mediated gene transfer of CSF-1 into op/op stromal cells to correct defective in vitro osteoclastogenesis

Colony-stimulating factor-1 (CSF-1) released by stromal cells in the bone microenvironment is essential for the proliferation of osteoclast progenitors. In op/op mutant mice, a thymidine insertion in the coding sequence of the CSF-1 gene results in CSF-1 deficiency that in turn leads to decreased osteoclast production and osteopetrosis. Because the osteopetrotic defect is due to the failure of stromal cells to produce CSF-1, we determined if retroviral-mediated gene transfer of the wild-type CSF-1 cDNA into op/op stromal cells would restore their ability to support osteoclast formation in vitro. A retroviral vector, L-CSF-1-SN, was constructed by inserting 1,867 bp of the wild-type CSF-1 cDNA into pLXSN. After transduction with L-CSF-1-SN or LXSN constructs, a stable PA317 packaging cell line that produced a high viral titre was isolated. Viral supernatant from this line was used to infect op/op bone marrow stromal cells. Stable L-CSF-1-SN op/op stromal clones overexpressed CSF-1 mRNA and released CSF-1 into conditioned medium, compared with no CSF-1 released by LXSN op/op stroma. The amount of CSF-1 produced by two clones was similar to the physiologic level released by normal littermate stroma. Southern blot analysis confirmed the presence of intact proviral sequences in transduced cells. In coculture assays, L-CSF-1-SN, but not LXSN, op/op stromal cells supported the formation of TRAP-positive multinucleated cells in the absence of exogenous CSF-1. These findings indicate that genetically engineered stromal cells may be used to improve defective osteoclastogenesis and suggest that targeting stromal cells to bone is a potentially useful therapeutic modality for treating bone disorders. J. Cell. Physiol. 176:323–331, 1998. © 1998 Wiley-Liss, Inc.     

The effective method of gene transfer into mammalian cells cultured in vitro

Efficiency of transformation by a number of vectors with the different selective markers of a set of cell lines has been studied for three different methods based on using calcium phosphate, polybrene, or electroporation. Electroporation is shown to be the most efficient one. Using this method with the system rat2k-cells-pAGO vector we have obtained the frequencies of transformation up to 2-3.10(-3). We suggest to use this system as a model for investigation of homologous recombination in the framework of the gene therapy project.     

Use of embryonic and somatic cells for production of transgenic domestic animals

In contrast to the highly developed genetic modification systems available for manipulating the mouse genome, at this time only simple gain of function modifications can be undertaken in domestic species. Clearly, the greatest barrier to gene targeting in domestic species has been the unavailability of cell lines that can be modified in vitro and still be used to generate a living organism. In the mouse, the embryonic stem (ES) cells and embryonic germ (EG) cells have fulfilled that role. While the nuclear transfer procedures have solved this problem in sheep and cattle, in swine ES and EG cells are still needed. In addition, targeting in domestic species is affected by the need to develop targeting constructs containing isogenic DNA regions. As a result, it is necessary to isolate the gene of interest, sequence required regions, and develop isogenic targeting constructs by technologies such as long-range PCR. On the positive side, enrichment protocols developed in the mouse can be applied to domestic species, thus facilitating the identification of correctly modified cell lines. Hence, progress in mammalian cloning, the development of EG cell lines, and advances in gene targeting presently allows the introduction of precise genetic modifications into the domestic animal genome.     

Gene transfer into chicken embryos as an effective system of analysis in developmental biology

Chicken embryos have been used as a model animal in developmental biology since the time of comparative and experimental embryology. Recent application of gene transfer techniques to the chicken embryo increases their value as an experimental animal. Today, gene transfer into chicken cells is performed by three major systems, lipofection, electroporation and the virus-mediated method. Each system has its own features and applicability. In this overview and the associated four minireviews, the methods and application of each system will be presented.     

Immune reconstitution in the immunodeficient chicken by transfer of embryonic lymphoid cells

Embryonic chickens were rendered immunodeficient by in ovo injection of homologous IgM on the 10th embryonic day. The immunodeficient embryos were intravenously given lymphoid cells taken from normal embryonic bursa, spleen or thymus on the 15th embryonic day. Gain of splenic or bursal weight, natural antibodies to sheep red blood cells (SRBC), frequencies of rosette forming cells (RFC) binding to SRBC or dinitrophenyl-SRBC (DNP-SRBC) and of immunoglobulin bearing cells (IBC) in the bursa and the spleen were investigated to assess the effect of transferred cells during the embryonic stage. Transferred bursal and splenic cells showed an ability to restore the frequency of RFC or IBC in the recipients. However, reversion from the immunodeficient state was not observed in the thymic cell transfer. These findings suggested that the cells derived from embryonic bursa and spleen contained stem cells which developed into RFC and also into precursors of IBC.     

Beyond the beneficial effects of translocations as an effective tool for the genetic restoration of isolated populations

Translocations are becoming increasingly popular as appropriate management strategies for the genetic restoration of endangered species and populations. Although a few studies have shown that the introduction of novel alleles has reversed the detrimental effects of inbreeding over the short-term (i.e., genetic rescue), it is not clear how effective such translocations are for both maintaining neutral variation that may be adaptive in the future (i.e., genetic restoration) and increasing population viability over the long-term. In addition, scientists have expressed concerns regarding the potential genetic swamping of locally adapted populations, which may eliminate significant components of genetic diversity through the replacement of the target population by the source individuals used for translocations. Here we show that bird translocations into a wild population of greater prairie-chickens (Tympanuchus cupido pinnatus) in southeastern Illinois were effective in both removing detrimental variation associated with inbreeding depression as well as restoring neutral genetic variation to historical levels. Furthermore, we found that although translocations resulted in immediate increases in fitness, the demographic recovery and long-term viability of the population appears to be limited by the availability of suitable habitat. Our results demonstrate that although translocations can be effective management tools for the genetic restoration of wild populations on the verge of extinction, their long-term viability may not be guaranteed unless the initial conditions that led to most species declines (e.g., habitat loss) are reversed.     

体外诱导鸡胚胎生殖细胞分化为神经干细胞

本研究探讨体外诱导鸡胚胎生殖细胞(EGCs)分化为神经干细胞(NSCs)的可能性.EGCs经类胚体(EB)阶段,以维生素A酸(RA)等进行诱导,在NSCs选择性培养基中筛培养扩增7 d,观察形态变化;采用RT-PCR法检测nestin基因表达及免疫细胞化学法检测nestin等NSCs特异性标志物,并对其扩增及分化能力进行观察.结果显示:EGCs经初级诱导,NSCs选择性培养基筛选培养7 d后,形成大量神经球样结构,可扩增传代;绝大部分神经球样结构呈nestin抗原阳性,表达nestin基因,且可分化为神经上皮样及少突胶质细胞.研究结果表明:RA等诱导的EGCs,经选择性培养基筛选培养可获得NSCs,有望为眼部神经变性疾病的治疗提供新的技术参考.     

A study of the efficiency of different methods to transfer a reporter gene to chicken embryonic cells

The methods of transfection of a plasmid with a reporter gene involving DNA injection into chicken embryonic cells were studied. The parameters of the efficient transfection of chicken blastodermal cells with a foreign gene have been determined (20–24 and up to 40% in culture and embryos, respectively). A high efficiency of transfection of primordial germ cells isolated from the gonads has been obtained after DNA injection into the dorsal aorta of 2.5-day-old chicken embryos.     

Bacterial gene transfer by natural genetic transformation in the environment.

Natural genetic transformation is the active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information. Since the famous discovery of transformation in Streptococcus pneumoniae by Griffith in 1928 and the demonstration of DNA as the transforming principle by Avery and coworkers in 1944, cellular processes involved in transformation have been studied extensively by in vitro experimentation with a few transformable species. Only more recently has it been considered that transformation may be a powerful mechanism of horizontal gene transfer in natural bacterial populations. In this review the current understanding of the biology of transformation is summarized to provide the platform on which aspects of bacterial transformation in water, soil, and sediments and the habitat of pathogens are discussed. Direct and indirect evidence for gene transfer routes by transformation within species and between different species will be presented, along with data suggesting that plasmids as well as chromosomal DNA are subject to genetic exchange via transformation. Experiments exploring the prerequisites for transformation in the environment, including the production and persistence of free DNA and factors important for the uptake of DNA by cells, will be compiled, as well as possible natural barriers to transformation. The efficiency of gene transfer by transformation in bacterial habitats is possibly genetically adjusted to submaximal levels. The fact that natural transformation has been detected among bacteria from all trophic and taxonomic groups including archaebacteria suggests that transformability evolved early in phylogeny. Probable functions of DNA uptake other than gene acquisition will be discussed. The body of information presently available suggests that transformation has a great impact on bacterial population dynamics as well as on bacterial evolution and speciation.     

Respiration and heat production in the embryonic development of chickens

A marked increase in the rate of oxagen consumption and heat production, measured by the direct method, is observed in the chick embryogenesis. The intensity of respiration and heat production of the embryos decreases as the development proceeds. During development the data obtained by direct and indirect calorimetry diverge. This divergence, referred to as psi u-function, gradually decreases by the moment of hatching. Differences in the value of heat production and psi u-function were found in crosses of meat and egg directions, related to differences in the growth rate and productivity of adult fowl.     

Modified electroporation protocol for Lactobacilli isolated from the chicken crop facilitates transformation and the use of a genetic tool

Isolates of Lactobacillus spp. from a collection of potentially probiotic strains isolated from the crops of broiler chickens were found to be non-electrotransformable using published techniques. One strain of Lactobacillus salivarius was shown to develop electrocompetence when an overnight culture was incubated in fresh medium. The effect was enhanced if glycine was incorporated into the fresh growth medium. When these modifications were applied to a number of other crop isolates of Lactobacillus spp., electrocompetence could be detected in approximately half the strains tested. Two temperature sensitive plasmid vectors that had been used for the genetic modification of other lactic acid bacteria were introduced into a crop strain of Lb. salivarius. Both showed temperature sensitivity at 42 °C and above but were relatively stable at 37 °C. The genetic tool harbouring an IS element allowed the delivery of the plasmid to multiple independent sites in the host chromosome. Harnessing such genetic tools will facilitate the future genetic analysis of the host bacterium.     

Functional conservation between rodents and chicken of regulatory sequences driving skeletal muscle gene expression in transgenic chickens

Background  

Regulatory elements that control expression of specific genes during development have been shown in many cases to contain functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental pattern. An example of such a module has been identified at the rat myosin light chain (MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated muscle gene expression in the mouse, would drive expression in similar domains in the chicken.     

Characterization of the ICSI-mediated gene transfer method in the production of transgenic pigs

Understanding the behavior of transgenes introduced into oocytes or embryos is essential for evaluating the methodologies for transgenic animal production. We investigated the expression pattern of a transgene transferred to porcine eggs by intracytoplasmic sperm injection‐mediated gene transfer (ICSI‐MGT) or pronuclear microinjection (PN injection). The introduction of the EGFP gene by ICSI‐MGT yielded significantly more embryos with non‐mosaic transgene expression (P < 0.01). In the ICSI‐MGT group, 61.5% (24/39) of the embryos were EGFP‐positive in all their component blastomeres at the morula stage, while fewer than 10% of such embryos were EGFP‐positive in the PN‐injection group. Using three types of transgenes, ranging from 3.0 to 7.5 kb in size, we confirmed that approximately one in four fetuses obtained by ICSI‐MGT was transgenic, suggesting that ICSI‐MGT is a practical method for transgenic pig production. Southern blot analysis of 12 transgenic fetuses produced by ICSI‐MGT revealed that the number of integrated transgene copies varied from 1 to 300, with no correlation between transgene size and the number of integrated copies. Fluorescence in situ hybridization analysis revealed that the transgenes were randomly integrated into a single site on the host chromosomes. Together, these data indicate that multiple‐copy, single‐site integration of a transgene is the primary outcome of ICSI‐MGT in the pig and that ICSI‐MGT is less likely than PN injection to cause transgene integration in a mosaic manner. Mol. Reprod. Dev. 79: 218–228, 2012. © 2011 Wiley Periodicals, Inc.     

Random-priming in vitro recombination: an effective tool for directed evolution.

A simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences is reported. The method involves priming template polynucleotide(s) with random-sequence primers and extending to generate a pool of short DNA fragments which contain a controllable level of point mutations. The fragments are reassembled during cycles of denaturation, annealing and further enzyme-catalyzed DNA polymerization to produce a library of full-length sequences. Screening or selecting the expressed gene products leads to new variants with improved functions, as demonstrated by the recombination of genes encoding different thermostable subtilisins in order to obtain enzymes more stable than either parent.     

Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropri-ate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to pro-duce mammary gland bioreactors.We established an effi-cient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was con-structed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.     

Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropriate post-translational modifications. The nuclear transfer of transgenic somatic cells is a powerful method to produce mammary gland bioreactors. We established an efficient gene transfer and nuclear transfer approach in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer and some of reconstructed embryos could develop into blastocyst in vitro. __________ Translated from Hereditas (Beijing), 2006, 28(12): 1513–1519 [译自: 遗传]