Increased Efficiency of Transgenic Livestock Production

Production of transgenic livestock by pronuclear microinjection of DNA into fertilized zygotes suffers from the compounded inefficiencies of low embryo survival and low integration frequencies of the injected DNA into the genome. These inefficiencies are one of the major obstacles to the large-scale use of pronuclear microinjection techniques in livestock. We investigated exploiting the properties of recombinase proteins that allow them to bind DNA to generate transgenic animals via pronuclear microinjection. In theory, the use of recombinase proteins has the potential to generate transgenic animals with targeted changes, but in practice we found that the use of RecA recombinase-coated DNA increases the efficiency of transgenic livestock production. The use of RecA protein resulted in a significant increase in both embryo survival rates and transgene integration frequencies. Embryo survival rates were doubled in goats, and transgene integration was 11-fold higher in goats and three-fold higher in pigs when RecA protein-coated DNA was used compared with conventional DNA constructs without RecA protein coating. However, a large number of the transgenic founders generated with RecA protein-coated DNA were mosaic. The RecA protein coating of DNA is straightforward and can be applied to any species and any existing microinjection apparatus. These findings represent significant improvements on standard pronuclear microinjection methods by enabling the more efficient production of transgenic livestock.     

Transgene delivery via intracellular electroporetic nanoinjection

Development of an effective cytoplasmic delivery technique has remained an elusive goal for decades despite the success of pronuclear microinjection. Cytoplasmic injections are faster and easier than pronuclear injection and do not require the pronuclei to be visible; yet previous attempts to develop cytoplasmic injection have met with limited success. In this work we report a cytoplasmic delivery method termed intracellular electroporetic nanoinjection (IEN). IEN is unique in that it manipulates transgenes using electrical forces. The microelectromechanical system (MEMS) uses electrostatic charge to physically pick up transgenes and place them in the cytoplasm. The transgenes are then propelled through the cytoplasm and electroporated into the pronuclei using electrical pulses. Standard electroporation of whole embryos has not resulted in transgenic animals, but the MEMS device allows localized electroporation to occur within the cytoplasm for transgene delivery from the cytoplasm to the pronucleus. In this report we describe the principles which allow localized electroporation of the pronuclei including: the location of mouse pronuclei between 21 and 28 h post-hCG treatment, modeling data predicting the voltages needed for localized electroporation of pronuclei, and data on electric-field-driven movement of transgenes. We further report results of an IEN versus microinjection comparative study in which IEN produced transgenic pups with viability, transgene integration, and expression rates statistically comparable to microinjection. The ability to perform injections without visualizing or puncturing the pronuclei will widely benefit transgenic research, and will be particularly advantageous for the production of transgenic animals with embryos exhibiting reduced pronuclear visibility.     

哺乳动物克隆的研究进展与应用前景

显微注射技术本身固有的缺点在很大程度上限制了转基因动物的研究及应用。近年来,体细胞基因打靶和体细胞克隆的最新研究进展表明,这两种技术相结合将成为制备大型转基因动物的有效途径。     

The use of recombinase proteins to generate transgenic large animals

The endogenous properties of recombinase proteins allow them to associate with and bind DNA to catalyze homologous recombination. These endogenous properties of cellular recombination enzymes may be useful to the field of transgenesis. The production of transgenic animals, in particular livestock, is an inefficient process by both conventional pronuclear microinjection techniques and nuclear transfer. Furthermore, the use of pronuclear microinjection is currently limited to the random addition of genes and does not allow for the replacement of an endogenous gene with a more desired one. The functions of cellular recombination enzymes have been exploited to develop a technique that is compatible with pronuclear microinjection and may make the process of generating transgenic livestock more efficient while also enabling the targeting of homologous chromosomal genes. In our hands, transgenic animals generated by the pronuclear microinjection of various recombinase protein-coated DNA fragments led to a higher than expected birth rate as well as transgene integration frequency. Most founder animals generated were likely mosaic, indicating that integration occurred after cell division. The presence of multiple related genes makes detection of any recombination event difficult. Overall, this technique is a straightforward, rapid, and efficient procedure that can be applied to any segment of DNA and any microinjection apparatus, and is less labor intensive than nuclear transfer.     

Production of functional transgenic mice by DNA pronuclear microinjection

Successful experiments involving the production of transgenic mice by pronuclear microinjection are currently limited by low efficiency of random transgene integration into the mouse genome. Furthermore, not all transgenic mice express integrated transgenes, or in other words are effective as functional transgenic mice expressing the desired product of the transgene, thus allowing accomplishment of the ultimate experimental goal - in vivo analysis of the function of the gene or gene network. The purpose of this review is to look at the current state of transgenic technology, utilizing a pronuclear microinjection method as the most accepted way of gene transfer into the mouse genome.     

Efficiency of transgenic rat production is independent of transgene-construct and overnight embryo culture

The aim of the present work was to study factors affecting the efficiency of transgenic technology in rats. We investigated the possible effects of pronuclear microinjection of buffer or different DNA-constructs on survival and development of rat zygotes in vitro and in vivo as well as the influence of overnight culture of these embryos before transfer into pseudopregnant foster mothers. The survival rate of zygotes and their development to the two-cell and morula stage was not affected by pronuclear microinjection with different DNA-constructs or buffer. However, the development to the blastocyst stage was impaired. Nevertheless, there was no difference in blastocyst development between zygotes injected with DNA-constructs or with buffer. Neither was there a difference in cell number in in vitro cultured blastocysts resulting from pronuclear microinjection of a transgene compared with non-injected controls. The survival rate to term was about 30% irrespective of whether microinjected embryos were transferred immediately after microinjection or after overnight culture in vitro. However, a reduction in the survival to term was observed for non-injected zygotes when they were developed in vitro to the two-cell stage before transfer to a pseudopregnant female. The percentage of transgenic rats that resulted from microinjected zygotes was similar in all groups regardless of the DNA-construct used (2.7-10.0%). In conclusion, the main detrimental factor in the microinjection of rat zygotes is the introduction of solution in the pronucleus. Overnight culture of zygotes between microinjection and oviduct transfer does not decrease the efficiency of transgenic rat generation.     

Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures

Pronuclear injection is currently the most often used method to make transgenic animals, but in some animal species it is temporally restrictive due to difficulty in visualizing pronuclei. However, the injection of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection technique of polylysine complexed DNA into pronuclear stage zygotes is described. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-l-lysine (degree of polymerization=51). Effects on transgenic frequency of both the lysine to phosphate ratio of polylysine to DNA and DNA concentration were studied. About 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysine/DNA mixture having a lysine to phosphate ratio (L:P) of 11 microinjection positive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysine/DNA were detected using agarose gel electrophoresis at the conditions which produced transgenic mice. The presence of polylysine with construct DNA altered thein vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic animals using DNA and polylysine in the absence of any other signal protein suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.     

乳腺特异高表达人促红细胞生成素转基因奶山羊的制备

目的制备乳腺特异性高表达人促红细胞生成素（hEPO）转基因奶山羊。方法采用牛β-乳球蛋白基因（BLG）调控元件和hEPO全长编码序列基因组DNA构建真核表达载体,应用受精卵原核注射的方法制备hEPO转基因山羊。结果在原核注射获得的188头羔羊中,经Southern blot法检测有4头羊含有hEPO基因,其中3头为母羊,1头公羊于出生后20d死亡;3头转基因母羊hEPO基因的拷贝数分别为1、10、2;Western blot检测结果显示转基因羊乳中的hEPO分子质量为32kDa;MTT法检测结果表明,在泌乳10d的3只转基因羊乳汁中,每毫升乳汁中hEPO活性分别达到1.17×10^2IU、1.90×10^4IU、1.91×10^4IU。结论牛BLG能够调控hEPO基因在山羊乳腺中高表达,为实现其他药用蛋白在山羊乳腺中表达奠定了基础。     

Transgenic animal production and animal biotechnology

Considerable progress has been made in methods for production of transgenic livestock; beginning with pronuclear microinjection over 20 years ago. New methods, including the use of viral vectors, sperm-mediated gene transfer and somatic cell cloning, have overcome many of the limitations of pronuclear microinjection. It is now possible to not only readily make simple insertional genetic modifications, but also to accomplish, more complex, homozygous gene targeting and artificial chromosome transfer in livestock.     

Timing of DNA integration, transgenic mosaicism, and pronuclear microinjection

Selection of transgenic embryos prior to embryo transfer is a means to increase the efficiency of transgenic livestock production. Among transgenic reporters, cytoplasmic expression of green fluorescent protein (GFP) has features that make it ideal for transgenic embryo selection. The primary objective of this study was to assess cytoplasmic expression of a specially designed GFP reporter as a tool for transgenic bovine embryo selection. A second objective was to evaluate this reporter for studying transgenic mosaicism related to timing of integration of pronuclear microinjected DNA. Transgenic embryos produced by pronuclear injection showed a discrete pattern of GFP expression with clusters at 25, 50, and 100% of blastomeres expressing GFP. This pattern of mosaicism is interpreted to indicate that the integration of microinjected DNA occurred, not only at the pronuclear stage, but also in the subsequent cell divisions. Among the GFP-positive transgenic embryos, only in 21% did all the blastomeres show the green fluorescence. Using the fraction of positive blastomeres within an embryo, the timing of integration of microinjected DNA was estimated. The frequency of nonmosaic embryos expressing GFP is consistent with published germline transmission success rates of transgenic cattle derived from pronuclear microinjected embryos. These results indicate the possible application of GFP as a marker of transgenic embryos and graphically illustrate underlying complexities in DNA integration in embryos subjected to pronuclear microinjection.     

Production of transgenic mice from vitrified pronuclear-stage embryos.

Cryopreservation of pronuclear-stage embryos would be useful for transgenic technology and genome preservation purposes. We compared a novel vitrification technique (solid surface vitrification, SSV) with another vitrification method in straws for cryosurvival and to generate transgenic progeny from cryopreserved mouse zygotes following microinjection. The SSV solution consisted of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4 M trehalose in M2 supplemented with 4 mg/ml BSA; the in straw vitrification solution was 7 M EG in M2 plus BSA. In experiment I, we compared the effect of the vitrification solutions alone, without cooling. No reduction was detected in survival and cleavage rates. In experiment II, SSV yielded a significantly higher percentage of morphologically normal zygotes (96%) that also cleaved at significantly higher rates (80%) when compared to that following "in straw" vitrification (68 and 66%, respectively). Cleavage rate in the non-vitrified control group (93%) was significantly higher than that of both vitrified groups. Following embryo transfer, there was no difference in the rate of pups obtained from the SSV, "in straw" vitrified, and control groups (97/457, 21%; 15/75, 20% and 56/209, 27%, respectively). In experiment III, SSV vitrified and fresh embryos were used for pronuclear DNA injection. Survival rate of vitrified embryos after microinjection was reduced compared to nonvitrified ones (64 vs. 72%, respectively; P < 0.05); however, development to two-cell stage was not different (76 vs. 72%, respectively). Following embryo transfer of vitrified vs. fresh microinjected embryos, in both cases 10% live pups were generated, including transgenic pups. The results demonstrated that the efficiency of generating transgenic pups from SSV vitrified pronuclear zygotes is comparable to that from fresh embryos.     

A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection

Creating transgenic mice is an important technology for genetic studies and is currently performed by pronuclear microinjection of plasmid DNA into fertilized eggs. Since survival of injected embryos and integration of plasmid DNA are not efficient, total efficiency is only around 3% with a standard protocol. To circumvent this problem, here we describe a novel transgenesis method, the Tol2-mediated cytoplasmic injection method (Tol2:CI). We injected a foreign DNA cloned in a Tol2-transposon vector together with the transposase mRNA into the cytoplasm of fertilized eggs. As expected, the survival rate of the injected embryos was increased drastically. Also, the foreign DNA was transposed from the plasmid to the genome and transmitted to the next generation very efficiently. Together, the overall transgenic efficiency became more than 20%. Considering its simplicity and perfect compatibility with existing pronuclear microinjection facilities, we propose that the Tol2:CI method is applicable to high throughput functional genomics studies.     

Advances in transgenic rat production

Predictable and reproducible production of transgenic rats from a standardized input of egg donors and egg recipients is essential for routine rat model production. In the course of establishing a transgenic rat service, transgenic founders were produced from three transgenes in outbred Sprague-Dawley (SD) rats and four transgenes in inbred Fischer 344 (F344) rats. Key parameters that affect transgenesis efficiency were assessed, including superovulation treatments, methods to prepare pseudopregnant recipients, and microinjection technique. Five superovulation regimens were compared and treatment with 20 IU PMSG and 30 IU HCG was selected for routine use. Four methods to prepare pseudopregnant egg recipients were compared and estrus synchronization with LHRHa and mating to vasectomized males was selected as most effective. More than 80% of eggs survived microinjection when modified pronuclear microinjection needles and DNA buffers were used. The efficiencies of transgenic production in rats and C57BL/6J (B6J) mice were compared to provide a context for assessing the difficulty of transgenic rat production. Compared to B6J mice, SD rat transgenesis required fewer egg donors per founder, fewer pseudopregnant egg recipients per founder, and produced more founders per eggs microinjected. Similar numbers of injection days were required to produce founders. These results suggest that SD rat transgenesis can be more efficient than B6J mouse transgenesis with the appropriate technical refinements. Advances in transgenic rat production have the potential to increase access to rat models.     

The Transgenic Rabbit as Model for Human Diseases and as a Source of Biologically Active Recombinant Proteins

Until recently, transgenic rabbits were produced exclusively by pronuclear microinjection which results in additive random insertional transgenesis; however, progress in somatic cell cloning based on nuclear transfer will soon make it possible to produce rabbits with modifications to specific genes by the combination of homologous recombination and subsequent prescreening of nuclear donor cells. Transgenic rabbits have been found to be excellent animal models for inherited and acquired human diseases including hypertrophic cardiomyopathy, perturbed lipoprotein metabolism and atherosclerosis. Transgenic rabbits have also proved to be suitable bioreactors for the production of recombinant protein both on an experimental and a commercial scale. This review summarizes recent research based on the transgenic rabbit model.     

Strain-Dependent Differences in the Efficiency of Transgenic Mouse Production

Transgenic mouse production via pronuclear microinjection is a complex process consisting of a number of sequential steps. Many different factors contribute to the effectiveness of each step and thus influence the overall efficiency of transgenic mouse production. The response of egg donor females to superovulation, the fertilization rate, egg survival after injection, ability of manipulated embryos to implant and develop to term, and concentration and purity of the injected DNA all contribute to transgenic production efficiency. We evaluated and compared the efficiency of transgenic mouse production using four different egg donor mouse strains: B6D2/F1 hybrids, Swiss Webster (SW) outbred, and inbred FVB/N and C57BL/6. The data included experiments involving 350 DNA transgene constructs performed by a high capacity core transgenic mouse facility. Significant influences of particular genetic backgrounds on the efficiency of different steps of the production process were found. Except for egg production, FVB/N mice consistently produced the highest efficiency of transgenic mouse production at each step of the process. B6D2/F2 hybrid eggs are also quite efficient, but lyze more frequently than FVB/N eggs after DNA microinjection. SW eggs on the other hand block at the 1-cell stage more often than eggs from the other strains. Finally, using C57BL/6 eggs the main limiting factor is that the fetuses derived from injected eggs do not develop to term as often as the other strains. Based on our studies, the procedure for transgenic mouse production can be modified for each egg donor strain in order to overcome any deficiencies, and thus to increase the overall efficiency of transgenic mouse production.     

No effect of recombinase-mediated DNA transfer on production efficiency of transgenic rats.

It was reported that recombinase-A protein (RecA)-coated exogenous DNA was more likely to be integrated into mouse, goat and pig genomes. The objective of this study was to investigate whether integration of exogenous DNA into the rat genome is improved by the recombinase-mediated DNA transfer. Pronuclear microinjection of RecA-coated EGFP or OAMB DNA resulted in a production efficiency of transgenic rats of 1.4-2.9%, comparable with 0.9-2.6% when non-coated control DNA was used. Intracytoplasmic injection of the sperm heads exposed to RecA-coated EGFP DNA did not produce any transgenic rats (0 vs. 0-2.8% in control groups). Thus, the recombinase-mediated DNA transfer contributed very little to the production of transgenic rats by means of pronuclear microinjection and intracytoplasmic sperm injection.     

动物转基因技术的新进展

到目前为止,原核注射是最可靠,也是使用最广泛的动物转基因方法.但该方法存在整合效率太低及不能定点整合的问题.在过去的20年里,出现了一些新的转基因方法,包括精子介导、反转录病毒介导、携带外源基因体细胞的核移植、ＥＳ细胞基因打靶技术等.但这些方法都未能根本地解决存在的问题.最近的一些文献中报道转基因技术在原有方法的基础上做出了改进后,取得了突破性进展.     

绿色荧光蛋白转基因小鼠模型的建立及胚胎冷冻保种

目的建立绿色荧光蛋白转基因小鼠模型,并采取胚胎冷冻的方法进行保种。方法通过原核显微注射法,把线性化、纯化后的外源基因pEGFP注射入BDF1小鼠受精卵中,胚胎移植给同期发情的假孕受体母鼠,获得子代小鼠。经鉴定对有表达的转基因鼠进行胚胎冷冻保种。结果移植注射胚胎385枚给30只假孕小鼠共出生了306只后代鼠,经PCR和southern blot检测得到5只阳性小鼠。F2代转基因鼠胚胎冷冻240枚胚胎。结论通过显微注射法使外源基因pEGFP在小鼠基因组中得到整合,建立了转pEGFP的转基因小鼠模型。     

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。     

转基因家兔模型制作方法

作为生物医学研究重要的实验动物模型,转基因家兔已经被广泛应用在人类心脑血管疾病、艾滋病以及癌症等生物医学研究领域,特别是利用转基因家兔模型在人类动脉粥样硬化实验研究中已经取得了令人注目的成绩。本文结合我们自己制作转基因家兔的经验、研究成果以及文献资料,详细介绍了利用原核显微注射法、直接将外源基因注入受精卵雄原核中的转基因家兔制作技术,回顾了利用转基因家兔模型在生物医学研究中取得的重要进展。