Germ cell transplantation in infertility mouse

This work investigated the spermatogenesis in an infertility BALB/c-nu mouse model by reinfusing germline stem cells into seminiferous tubules. Donor germ cells were isolated from male FVB/NJ-GFP trensgenic mice. Seminiferous tubule microinjection was applied to achieve intratubular germ cell transfer. The germ cells were injected into exposed testes of the infertility mice. We used green fluorescence and DNA analysis of donor cells from GFP transgenic mice as genetic marker. The natural mating and Southern blot methods were applied to analyze the effect of sperm cell transplantation and the sperm function after seminiferous tubule microinjection. The spermatogenesis was morphologically observed from the seminiferous tubules in 41/60 （68.33%） of the injected recipient mice using allogeneic donor cells. In the colonized testes, matured spermatozoa were seen in the lumen of the seminiferous tubules. In this research, BALB/c-nu infertility mouse model, the recipient animal, was used to avoid immunological rejection of donor cells, and germ cell transplantation was applied to overcome infertility caused by busulfan treatment. These results demonstrate that this technique of germ cell transplantation is of great use. Germ cell transplantation could be potentially valuable to oncological patients.     

Advances in mammalian spermatogonial stem cell transplantation

Spermatogonial stem cell (SSC) transplantation is a novel technique by which testicular cells from normal, transgenic or mutant donor are introduced into the seminiferous tubules of recipient testes through microinjection. Subsequently, donor SSCs survive,migrate, anchor and proliferate in the recipient testis, furthermore, initiate spermatogenesis and even produce sperms capable of fertilization. This technique provides a new approach for the researches of spermatogenesis mechanism, regeneration of spermatogenesis in sterile individuals and reproduction of transgenic animals. This review focuses on the methodological breakthroughs and highlights the recent findings that have substantially increased understanding of SSC biology. The article provides a comprehensive overview of this technique and its multiple applications in basic science and medicine. And the perspective direction of this field in the near future is proposed.     

原核显微注射产生转基因绵羊胚胎

体外采集绵羊卵丘卵母细胞复合体,成熟培养24 h,经过体外受精培养17 h,比较高速离心对胚胎发育的影响;高速离心可以使黑色脂滴甩到一边从而使受精卵原核清晰可见.然后将绵羊乳腺特异表达人肝细胞再生增强因子和真核细胞表达增强绿色荧光蛋白(Enhanced Green fluorescence protein,EGFP)的载体DNA显微注射于绵羊受精卵雄原核中,并将异构胚在SOF液中发育培养.结果表明:高速离心组囊胚率低于对照组,但是没有显著性差异(P》0.05);显微注射外源基因2天后在激光共聚焦显微镜下可见荧光胚胎;PCR检测5个荧光胚胎均可见特异性条带.在原核显微注射生产转基因胚胎中,绿色荧光蛋白可作为标记基因进行早期胚胎筛选,为提高转基因动物移植效率奠定实验基础.     

Nuclear transplantation in different strains of zebrafish

Single later blastula nuclei from AB strain of zebrafish (Danio rerio) were transplanted into enucleated unfertilized eggs of Long fin strain. Of 1119 cloning embryos, 14 reconstructed embryos developed into fry. DNA fingerprinting systems of the cloned fish were similar to those of the nuclear donor fish, but were distinctly different from those of the nuclear recipient fish. It confirmed that the genetic material originated from nuclear donor cell other than from nuclear recipient egg. The research suggested that the basic technique for nuclear transplantation performed with different strains of zebrafish has made a breakthrough. It should be helpful for the study of some important developmental problems such as gene function, the regulation ogene expression during animal development, the developmental potential of a nucleus and the interactions between the donor nucleus and the recipient cytoplasm, etc.     

Expression of human erythropoietin directed by mWAP promoter in mammary gland of transgenic mice

The present work has generated transgenic mice with a hybrid gene construct consisting of genomic sequences encodinghuman erythropoietin (hEPO) and governed by regulatory sequences of mousewhey acidic protein (mWAP). The construct proved effective by transient expression in lactating animal. After introducing hybrid gene construct into single-cell embryo via pronuclear microinjection, surviving embryo are reimplanted into pseudopregnant foster mother mouse. 58 mice of 86 generation zero mice obtained were identified to be positive by PCR-Southern blot and genomic DNA Southern blot methods. The integration rate is 67%.hEPO was expressed in the milk of 16 mice of 39 mice measured byhEPO ELISA kit The expression level gets over 15 μg/mL.     

Production of transgenic calves by somatic cellnuclear transfer

Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector(pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant(Neo^r) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was cartied out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves,and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves.PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.     

Reconstitution with syngeneic plus allogeneic or xenogeneic bone marrow leads to specific acceptance of allografts or xenografts

Clinical organ transplantation between genetically disparate individuals currently requires the use of chemotherapeutic agents to suppress the rejection reaction. The deleterious side effects of these reagents and their inability to prevent rejection completely has led to a continuing search for methods to induce specific transplantation tolerance in adult recipients. Numerous experimental animal models utilizing irradiation and bone marrow transplantation coincident with organ transplantation have been proposed. Bone marrow transplantation, however, has its own major complications, including graft-versus-host reactions and immunoincompetence, probably resulting from a failure of appropriate immune cell interactions in the reconstituted host. We have now attempted to overcome these difficulties by reconstituting the irradiated host with T-cell depleted bone marrow containing both host (syngeneic) and donor (allogeneic or xenogeneic) components. This technique leads to long-term survival of the reconstituted animals and specific prolongation of subsequent skin grafts of donor type. Animals reconstituted in this fashion are fully reactive to third-party allografts and xenografts and do not appear to manifest signs of graft-versus-host disease.     

Monoclonal antibodies to promote marrow engraftment and tissue graft tolerance

Allogeneic reactions are the major limitation to organ transplantation. These are manifested as rejection of the grafted tissue, and also, in the case of bone marrow transplantation (BMT), graft-versus-host disease (GVHD). Recent methods of avoiding GVHD, by depleting T cells from donor marrow, have led to an increased incidence of marrow graft rejection. Current recipient conditioning protocols involving drugs or irradiation cannot safely be increased, so alternatives must be found. Monoclonal antibodies can be used to control immune responses in vivo, and would be useful in this context if we could define and deplete the cells responsible for marrow rejection. We show here that elimination of residual L3T4+ and Lyt-2+ cells from mice receiving fully mismatched bone marrow abrogates rejection and promotes tolerance to donor-type skin grafts, even in sub-lethally irradiated recipients.     

哺乳动物克隆的现状和研究进展

 哺乳动物细胞克隆是20世纪末生命科学领域最引人注目的高新技术,该技术对于优良种畜的复制、减少试验用动物数目、动物遗传多样性保存及濒危动物挽救、转基因动物培育等方面具有重要意义。近年来克隆技术发展迅速,多种哺乳动物相继克隆成功,但也存在克隆效率太低、克隆动物表型正常而实质异常的问题。本文详细阐述了克隆效率太低、克隆动物表型正常而实质异常问题,介绍了当前动物克隆技术的发展现状,并对动物克隆涉及的技术进行了总结和概括,着重介绍了卵母细胞的去核方法和重组胚的构建方法。     

转基因动物的应用与展望

转基因动物是生物工程中一个新兴领域．它在基因表达与调控的基础理论研究、贵重药物生产、建立人类疾病模型动物、生产供人类移植用器官、培育家畜新品种等多方面得到应用，并显示出极优越的应用前景。     

基因表达系统与转基因动物乳腺反应器

出于研究、医疗或工业目的，常常需要大量置备各种生物活性蛋白质，为此已经建立了多种基因工程表达系统，如微生物发酵系统、真核细胞培养系统、转基因植物表达系统和转基因动物表达系统等。近几年，利用转基因动物作为生物反应器由乳汁中生产蛋白药物的研究取得了很大进展，有多种动物可被用于转基因。基本过程是将基因构件显微注射到单细胞期受精卵，基因构件以一定几率整合到受体基因组中。通常转基因和其表达模式可忠实地遗传。许多蛋白将以高浓度低成本由转基因家畜的奶中生产。这些转基因动物与传统的动物细胞培养和细菌发酵技术相比尤显高效。乳腺能完成包括二硫桥形成、酰胺化、羧基化和糖基化在内的翻译后修饰，１头转基因羊就是１套发酵罐。据预测，到２０１０年由转基因动物生产的蛋白药物占全部基因工程药物的比率将增长到９５％以上。     

转基因动物研究概述

本文主要对转基因动物的研究进展及其常用的各种转基因方法、转基因动物的应用和研究前景进行概述，分析了当前制约转基因动物发展的主要因素，而加强相关基础理论的研究和克服转基因技术的瓶颈将会大大加快转基因动物发展的步伐。     

滨海湿地鸟类的动物生态与保护生物学研究

采用生态学研究方法和分子生物学技术,以滨海湿地鸟类等脊椎动物为研究对象,尤其以鹭科鸟类为主,研究动物多样性与环境的相互关系,分析鸟类繁殖生态及其关键环境影响因素,探讨滨海湿地鸟类食物链的污染物转移与富集规律,建立鸟类物种及性别的分子生物学鉴定技术,分析鹭科鸟类种群遗传多样性及其影响因素,探讨濒危物种的致危机制,以期促进生物多样性及其栖息地的保护.     

小鼠胚胎移植技术优化及对巨细胞病毒净化研究

目的 建立小鼠巨细胞病毒净化方法,以获得无MCMV感染的小鼠。方法 利用胚胎移植技术,通过使用不同激素、不同发情周期注射激素、受体鼠品系、不同的移植方法、不同时期胚胎移植,以及移植胚胎数量等对比试验,优化了净化条件。利用优化的胚胎移植净化方法,对供体鼠进行了净化。结果 SIGMA生产的激素,在10 IU剂量下超排得到的可用胚胎数量约为17枚/只;在小鼠发情间期超排得到的可用胚胎最多,超排的可用胚胎数约为23枚/只;选取C57雄性小鼠与ICR雌性小鼠交配的子一代作为受体鼠,产仔率达44.5%;输卵管移植较子宫移植效果好,产仔率为40.64%;移植2细胞胚胎的妊娠率为80%,明显优于单细胞和8细胞;受体鼠移植24枚胚胎时,产仔率达到了43.75%。利用优化的小鼠巨细胞病毒胚胎移植净化方法,净化得到了无MCMV感染的小鼠。结论 建立了小鼠巨细胞病毒净化方法,为获得无MCMV感染的小鼠种群提供了可靠的保障。     

Genome editing and animal models

Transgenic technology allows a gene of interest to be introduced into the genome of a laboratory animal, and provides an extremely powerful tool to dissect the molecular mechanisms of disease. Transgenic mouse models made by microinjection of DNA into zygotic pro- nuclei in particular have been widely used by the genetics community for 30 years. However, it remains a rather crude approach： injected sequences randomly insert in multiple copies as concatamers, they can be mutagenic, and they have variable or silenced expression depending on the site of integration, a phenomenon called position effects. As a result, multiple lines are required in order to confirm appropriate transgene expression. This can be partially overcome by flanking transgenes with insulator sequences to protect the transgene from the influence of the sur- rounding regulatory elements. Large （〈300 kb） BAC- based transgenic vectors have also been shown to be more resistant to position effects. However, animals carrying extra copies of fairly large regions of the genome could have unpredictable phenotypes. The most effective method used to control for position effects is to target transgene insertion to specific genomic loci, the so-called targeted transgenesis; for instance, the fast, site-specific transgenic technology TargattTM. The purpose of this review is to provide an overview on the current existing methods for making targeted transgenic mouse models.     

Green fluorescent protein （GFP） transsenic pig produced by somatic cell nuclear transfer

Transgenic somatic cell nuclear transfer is a very promising route for producing transgenic farm animals. Research on GFP transgenic pigs can provide useful information for breeding transgenic pigs, human disease models and human organ xenotransplantation. In this study, a liposomal transfecUon system was screened and transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes. The development of reconstructed embryos both in vitro and in vivo was observed, and GFP expression was determined. The results showed that porcine fe- tal-derived fibroblast cells cultured with 4.0 μL/mL liposome and 1.6 μg/mL plasmid DNA for 6 h resulted in the highest transfecUon rate （3.6%）. The percentage of GFP reconstructed embryos that de- veloped in vitro to the blastocyst stage was 10%. Of those the GFP positive percentage was 48%. Reconstructed transgenic embryos were transferred to 10 recipients. 5 of them were pregnant, and 3 delivered 6 cloned piglets in which 4 piglets were transgenic for the GFP as verified by both GFP protein expression and GFP DNA sequence analysis. The percentage of reconstructed embryos that resulted in cloned piglets was 1.0%; while the percentage of piglets that were transgenic was 0.7%. This is the first group of transgenic cloned pigs born in China, marking a great progress in Chinese transgenic cloned pig research.     

Production of gene-targeted sheep by nuclear transfer from cultured somatic cells

It is over a decade since the first demonstration that mouse embryonic stem cells could be used to transfer a predetermined genetic modification to a whole animal. The extension of this technique to other mammalian species, particularly livestock, might bring numerous biomedical benefits, for example, ablation of xenoreactive transplantation antigens, inactivation of genes responsible for neuropathogenic disease and precise placement of transgenes designed to produce proteins for human therapy. Gene targeting has not yet been achieved in mammals other than mice, however, because functional embryonic stem cells have not been derived. Nuclear transfer from cultured somatic cells provides an alternative means of cell-mediated transgenesis. Here we describe efficient and reproducible gene targeting in fetal fibroblasts to place a therapeutic transgene at the ovine alpha1(I) procollagen (COL1A1) locus and the production of live sheep by nuclear transfer.     

转基因玉米遗传转化的研究进展

转基因玉米是目前植物基因工程的研究热点之一,已有转基因抗虫玉米、抗除草剂玉米进入商品化生产。通过对转基因玉米遗传转化方法的研究,概括了目前在转基因玉米的遗传转化中所用的各种方法、及其优缺点,以及已取得的成果和遗传转化的检测分析方法的研究进展。     

外源基因在转基因动物乳腺中的特异性表达研究

动物基因工程研究最大的突破就是转基因动物研究的进展,自八十年代初第一批转基因小鼠问世以来,转基因动物的研究已从方法学研究步入了应用性研究阶段,转基因动物除作为研究工具广泛应用于发育生物学、免疫学、遗传学以及医学等生命科学领域外,外源基因在转基因动物的特异性表达,尤其是在乳腺的表达,又可将转基因用作生物反应器进行了生物活性蛋白的生产而于商业生产,在转基因动物乳腺的特异性表达研究中,寻找乳蛋白基因调控     

富钾植物DNA导入早稻变异后代的RAPD分析

应用花粉管通道法和浸种法将两种富钾植物空心莲子草和商陆DNA导入到不同的水稻品种中,结果在其后代产生了广泛的变异,并从中筛选出耐低钾的变异材料。应用PCR技术对其中耐低钾的4个变异材料进行了RAPD分析。结果表明,所用100个随机引物中有7个引物在受体和变异后代间扩增出了多态性产物。后代和受体的相似系数均在90%以上,而与供体的相似系数只有6%以下,其中有三个引物的扩增产物中出现了供体的特异性带。这说明空心莲子草和商陆DNA导入水稻后确实引起了水稻基因组结构的变化,同时也为变异后代所发生的生物学性状的改变提供了直接的分子证据。