体细胞介导制备转基因动物的新途径

体细胞介导制备转基因动物的技术路线是在核移植技术的基础上建立起来的。它的问世不过只有四五年时间 ,但显示了其具有广泛的应用前景。本综述比较了不同制备转基因动物技术的优缺点 ,介绍了体细胞介导制备转基因动物的基本技术路线 ,着重阐述了体细胞转染和整合细胞的克隆 ,以及体细胞介导的转基因技术的局限性和应用前景     

Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer

The production of animals by somatic cell nuclear transfer (SCNT) is inefficient, with approximately 2 % of micromanipulated oocytes going to term and resulting in live births. However, it is the most commonly used method for the generation of cloned transgenic livestock as it facilitates the attainment of transgenic animals once the nuclear donor cells are stably transfected and more importantly as alternatives methods of transgenesis in farm animals have proven even less efficient. Here we describe piggyBac-mediated transposition of a transgene into porcine primary cells and use of these genetically modified cells as nuclear donors for the generation of transgenic pigs by SCNT. Gene transfer by piggyBac transposition serves to provide an alternative approach for the transfection of nuclear donor cells used in SCNT.     

Expression of enhanced green fluorescent protein in porcine- and bovine-cloned embryos following interspecies somatic cell nuclear transfer of fibroblasts transfected by retrovirus vector

Interspecies somatic cell nuclear transfer (iSCNT) has emerged as an important tool for studying nucleo-cytoplasmic interactions and cloning of animals whose oocytes are difficult to obtain. This study was designed to explore the feasibility of employing transgenic fibroblasts as donor cells for iSCNT. The study examined the chromatin morphology, in vitro development, and expression of an enhanced green fluorescent protein (EGFP) gene in porcine- and bovine-cloned embryos produced by iSCNT of fetal fibroblast transfected with a pLNbeta-EGFP retroviral vector. Parthenogenetic and transfected or nontransfected intraspecies SCNT embryos were used as controls for comparison. Analysis of data revealed that xenogenic oocyte was able to reprogram somatic cells of different genus and supports their in vitro development to the blastocyst stage. However, the developmental rates of transgenic iSCNT embryos to the blastocyst stage were significantly lower than those of intraspecies SCNT embryos. The reduction in development rates was however, not due to integration of the transgene as the lower (P < 0.05) development rates of the intraspecies SCNT porcine or bovine embryos did not differ between transgenic and nontransgenic groups. Expression of EGFP was observed in 100% of blastocysts and mosaicism was not observed. Furthermore, after iSCNT of porcine or bovine donor nuclei into xenogenic ooplasm, patterns of nuclear remodeling in reconstructed embryos were similar. In conclusion, our data demonstrated the feasibility of producing transgenic iSCNT embryos. To our knowledge, this is the first report of transgenic cloned embryo production by iSCNT approach. In the future, this may provide a powerful research tool for studying developmental events in domestic animals and provide marked cell lines for other genetic manipulations.     

动物体细胞核移植技术的研究现状

动物体细胞核移植的成功表明动物体细胞的分化是可逆的,这是近年来人类在细胞生物学及发育生物学领域取得的最伟大的成就之一。１９９７年首例体细胞核移植绵羊在世界上诞生,之后不久出现了体细胞核移植小鼠及牛,并通过体细胞核移植技术制作了转基因动物。成就是不言而喻的,但其中也暴露出一些需克服和解决的问题。本文围绕世界上首例体细胞克隆羊诞生的背景、体细胞核移植技术目前的研究状况、体细胞核移植过程中的核质互作、体细胞核移植技术目前存在的问题、体细胞核移植技术在制作转基因动物上的应用等方面,扼要介绍了体细胞核移植技术在最近两年内取得的进展。     

供核细胞对体细胞核移植效率的影响

利用体细胞核移植技术克隆动物、生产转基因家畜具有极大的应用潜力。然而,核移植效率低下、克隆后代形态异常等问题仍然制约着体细胞核移植技术的产业化进展。影响体细胞核移植效率的因素很多,该文着重从供核细胞的类型、细胞体外培养、细胞凋亡及转基因操作等方面阐述其对体细胞核移植效率的影响。     

Production of recombinant albumin by a herd of cloned transgenic cattle

Purified plasma derived human albumin has been available as a therapeutic product since World War II. However, cost effective recombinant production of albumin has been challenging due to the amount needed and the complex folding pattern of the protein. In an effort to provide an abundant source of recombinant albumin, a herd of transgenic cows expressing high levels of rhA in their milk was generated. Expression cassettes efficiently targeting the secretion of human albumin to the lactating mammary gland were obtained and tested in transgenic mice. A high expressing transgene was transfected in primary bovine cell lines to produce karyoplasts for use in a somatic cell nuclear transfer program. Founder transgenic cows were produced from four independent cell lines. Expression levels varying from 1–2 g/l to more than 40 g/l of correctly folded albumin were observed. The animals expressing the highest levels of rhA exhibited shortened lactation whereas cows yielding 1–2 g/l had normal milk production. This herd of transgenic cattle is an easily scalable and well characterized source of rhA for biomedical uses.     

野生动物种间体细胞核移植研究进展

种间体细胞核移植 (Interspecies somatic cell nuclear transfer, iSCNT) 作为体细胞核移植 (Somatic cell nuclear transfer, SCNT) 中极具潜力的研究方向之一,在转基因动物的生产、优良种群的繁育、濒危动物的保护、灭绝物种的“再生”以及野生动物繁育和生物多样性保护等方面具有广泛的应用前景。本文通过对猫科、犬科、牛科、猪科和西貒科等野生动物的种间核移植技术的研究进展和技术屏障进行阐述,为促进iSCNT技术在野生动物繁殖、育种和多样性保护等方面的进一步应用提供理论基础和技术指导。     

Global gene expression analysis of bovine blastocysts produced by multiple methods

Reproductive efficiency using somatic cell nuclear transfer (SCNT) technology remains suboptimal. Of the various efforts to improve the efficiency, chromatin transfer (CT) and clone-clone aggregation (NTagg) have been reported to produce live cloned animals. To better understand the molecular mechanisms of somatic cell reprogramming during SCNT and assess the various SCNT methods on the molecular level, we performed gene expression analysis on bovine blastocysts produced via standard nuclear transfer (NT), CT, NTagg, in vitro fertilization (IVF), and artificial insemination (AI), as well as on somatic donor cells, using bovine genome arrays. The expression profiles of SCNT (NT, CT, NTagg) embryos were compared with IVF and AI embryos as well as donor cells. NT and CT embryos have indistinguishable gene expression patterns. In comparison to IVF or AI embryos, the number of differentially expressed genes in NTagg embryos is significantly higher than in NT and CT embryos. Genes that were differentially expressed between all the SCNT embryos and IVF or AI embryos are identified. Compared to AI embryos, more than half of the genes found deregulated between SCNT and AI embryos appear to be the result of in vitro culture alone. The results indicate that although SCNT methods have altered differentiated somatic nuclei gene expression to more closely resemble that of embryonic nuclei, combination of insufficient reprogramming and in vitro culture condition compromise the developmental potential of SCNT embryos. This is the first set of comprehensive data for analyzing the molecular impact of various nuclear transfer methods on bovine pre-implantation embryos.     

Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle

The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear transfer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 μg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blastocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, respectively). The development rates to the blastocyst stage of NT embryos were significantly different among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal oviduct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo developmental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.     

转基因动物技术的研究进展及应用

文章综述了转基因动物的制作的主要方法及其优缺点,包括显微原核注射法、逆转录病毒感染法、胚胎干细胞介导法、体细胞核移植技术、精子载体法、胞浆内单精子注射法以及卵母细胞载体法等。阐述了转基因动物技术在人类疾病模型、生产人体器官、动物反应器和改良动物品种及其生产性能等方面的应用,并提出转基因动物技术存在的一些技术难题和安全性问题。     

体细胞核移植诱导的表观遗传学变化

体细胞核移植技术是指将一个分化的体细胞核置入去核的卵母细胞中,并发育产生与供体细胞遗传背景一致的克隆后代的技术。目前,世界上通过体细胞核移植技术已经产生了许多的克隆动物。但克隆过程中还存在着很多问题,比如,克隆效率太低、克隆个体常伴有表型异常和早亡等,从而使该技术应有的应用潜力不能得到充分的发挥。体细胞表观遗传学重编程的不完全或紊乱是造成核移植诸多问题的主要原因。近十多年来,人们对体细胞核移植后的重编程进行了广泛的研究,其核心内容包括核及核外结构的重塑、DNA甲基化模式的重建、基因印迹和x染色体失活、组蛋白乙酰化模式的重建、端粒长度恢复等,以期能够对其重编程加以人为干预,从而提高动物克隆效率。本文拟对体细胞核移植诱导的重编程研究进展加以综述,希望对体细胞重编程机制的阐明有所启发。     

Advances in livestock nuclear transfer

Cloning and transgenic animal production have been greatly enhanced by the development of nuclear transfer technology. In the past, genetic modification in domestic animals was not tightly controlled. With the nuclear transfer technology one can now create some domestic animals with specific genetic modifications. An ever-expanding variety of cell types have been successfully used as donors to create the clones. Both cell fusion and microinjection are successfully being used to create these animals. However, it is still not clear which stage(s) of the cell cycle for donor and recipient cells yield the greatest degree of development. While for the most part gene expression is reprogrammed in nuclear transfer embryos, all structural changes may not be corrected as evidenced by the length of the telomeres in sheep resulting from nuclear transfer. Even after these animals are created the question of "are they really clones?" arises due to mitochondrial inheritance from the donor cell versus the recipient oocyte. This review discusses these issues as they relate to livestock.     

Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle

Genetically modified animals have many poten-tial applications in basic research, human medicine and agriculture. Pronuclear DNA microinjection has been almost the only practical means of producing transgenic animals during the last 20 years, but the low efficiency (1%—5%)[1] of this method has actu-ally been the obstacle that hampered its further appli-cation in animal biotechnology. The birth of Dolly[2], the first somatically cloned animal, made it possible to produce transgenic animals b…     

DNA甲基化和组蛋白修饰在克隆动物发育过程中的作用

体细胞核移植在农业应用、生产疾病模型动物、转基因家畜或产生人胚胎干细胞来治疗人类的疾病方面有巨大的应用潜力。虽然已经成功克隆出多种哺乳动物, 但该技术仍存在一些未解决的问题, 包括产生克隆动物的效率低和克隆动物的异常等。异常的表观遗传重编程是克隆胚胎发育失败的一个重要因素。文章重点论述了DNA甲基化、组蛋白修饰及其与克隆胚胎发育的关系。了解表观遗传调控机制有助于解决核移植技术中存在的问题, 有利于更好地应用这项技术。     

Nuclear transfer: a new tool for reproductive biotechnology in cattle

Recent evolutions of somatic cloning by nuclear transfer are reported, especially in the bovine species where potential applications are underway for biomedicine in association with transgenesis, or for agriculture by improving livestock. The overall efficiency of this biotechnology remains low in terms of viable offspring, but significant progress has been achieved on the different steps of the technique. However, the in vivo development of bovine blastocysts derived from somatic nuclear transfer is characterised by some important features that lead to the "cloning syndrome". Important losses occur during the peri-implantation period and further late foetal loss is observed in association with the Large Offspring Syndrome. About 60-70% of the cloned calves born survive normally to the adult stage and present an apparently normal physiology. Recent data already available on bovine somatic clones of both sexes indicate that they have a zootechnical performance similar to non cloned animals and they are able to reproduce normally without the pathologies associated to cloning thus confirming that the deviations observed in clones are of epigenetic origin and not transmitted to the progeny.     

家畜转基因育种研究进展

转基因技术可以将外源基因导入家畜基因组,使其获得新的可遗传性状,为培育优良家畜品种提供了革命性途径。DNA显微注射法和体细胞克隆法是制备转基因家畜最常用的方法。应用转基因技术可以进行家畜抗病育种(抗病毒、抗菌和抗寄生虫),改良家畜的生产性状(胴体组成、奶品质、产毛、繁殖力和生长速度),培育环保型家畜新品种。文章从动物转基因技术入手,阐明其在家畜品种改良方面的研究现状和策略,并探讨家畜转基因育种面临的问题和应用前景。     

Gene targeting from laboratory to livestock: Current status and emerging concepts

The development of methods for cell-mediated transgenesis, based on somatic cell nuclear transfer, provides a tremendous opportunity to shape the genetic make-up of livestock animals in a much more directed approach than traditional animal breeding and selection schemes. Progress in the site-directed modulation of livestock genomes is currently limited by the low efficiencies of gene targeting imposed by the low frequency of homologous recombination and limited proliferative capacity of primary somatic cells that are used to produce transgenic animals. Here we review the current state of the art in the field, discuss the crucial aspects of the methodology and provide an overview of emerging approaches to increase the efficiency of gene targeting in somatic cells.     

Using a modified piggyBac transposon-combined Cre/loxP system to produce selectable reporter-free transgenic bovine mammary epithelial cells for somatic cell nuclear transfer

Transposon systems are widely used for genetic engineering in various model organisms. PiggyBac (PB) has recently been confirmed to have highly efficient transposition in the mouse germ line and mammalian cell lines. In this study, we used a modified PB transposon system mediated by PB transposase (PBase) mRNA carrying the human lactoferrin gene driven by bovine β-casein promoter to transfect bovine mammary epithelial cells (BMECs), and the selectable reporter in two stable transgenic BMEC clones was removed using cell-permeant Cre recombinase. These reporter-free transgenic BMECs were used as donor cells for somatic cell nuclear transfer (SCNT) and exhibited a competence of SCNT embryos similar to stable transgenic BMECs and nontransgenic BMECs. The comprehensive information from this study provided a modified approach using an altered PB transposon system mediated by PBase mRNA in vitro and combined with the Cre/loxP system to produce transgenic and selectable reporter-free donor nuclei for SCNT. Consequently, the production of safe bovine mammary bioreactors can be promoted.     

Production of goats by somatic cell nuclear transfer.

In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line.