Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

Background

Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals.

Results

The linker protein, a monoclonal antibody (mAb C), is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57) of transgenic pigs (F0 generation).

Conclusions

Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.     

Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system

Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

一种改进的显微注射用DNA片段的纯化方法

目的显微注射用DNA的纯度是影响转基因动物制备成功与否的重要因素,本文建立一种可行的适用于普通实验室的纯化DNA方法,替代普遍使用的试剂盒纯化方法。方法分别使用酚-氯仿多次抽提法及常规的凝胶提取试剂盒纯化含有蚓激酶基因的DNA片段,通过显微注射技术将纯化的DNA片段导入小鼠受精卵的原核,制备转基因小鼠。根据转基因实验的结果对两种方法进行比较。结果使用两种方法纯化DNA均能获得转基因小鼠。在DNA纯度及注射卵的存活率上,两种方法无明显差别;在移植卵的出生率及转基因阳性率上,抽提法优于试剂盒法。结论本实验建立的抽提方法可以替代试剂盒方法纯化显微注射用DNA片段,在降低实验成本、简化实验条件及提高转基因阳性率方面具有优势。     

Generation of transgenic mice by transfection of pronuclear embryos using lipid-DNA complexes

We had previously developed a methodology to introduce foreign DNA into mouse eggs and embryos using cationic lipids as vectors. In this report we use this technique to produce transgenic animals. Mouse embryos at the pronuclear stage were transfected using a mixture of a plasmid DNA, encoding for a nuclear form of beta-galactosidase, and a commercial lipid transfection reagent. Embryos were washed and incubated overnight. Those that cleaved and develop to the 2-cell stage of normal appearance were transferred to the Fallopian tubes of pseudopregnant foster mothers. We analysed a total of 158 offspring and found two, a female and a male, to be transgenic (1.27% of the total). Integration of the foreign DNA in the female was showed by Southern blot. Both animals expressed the lacz in several organs, but none of them either displayed expression in the germ cells or transmitted the transgene to their offspring. Taken together our results show that lipid transfection can generate transgenic mice, but the efficiency needs to be improved for this method to be widely applied.     

A comparative study on the integration of exogenous DNA into mouse, rat, rabbit, and pig genomes.

Transgenic mammals, from small laboratory rodents to domestic animals, have been successfully produced to date, but their production efficiency within or across species has been variable. This is probably due to the differences in the type of injected DNA and/or technical procedures employed in each laboratory, as well as the reproductive characteristics of the species. Here we report the direct comparison of the efficiencies of producing transgenic mice, rats, rabbits and pigs by one technician using a fusion gene composed of the bovine alpha S1-casein promoter and human growth hormone (hGH) gene. Before the fusion gene was injected into the zygotes, high magnitude centrifugation to visualize the pronuclei was necessary for all of the pig zygotes and one-third of the rabbit zygotes, but not for mouse and rat zygotes. Post-injection survival of the mouse zygotes (67.1%) was lower than those of the rat, rabbit and pig zygotes (89.6 to 100%). The volume change of the pronucleus following DNA injection was the lowest in mice (50% increase), moderate in rabbits (148% increase), and the most prominent in rats (238% increase). The data from only 1 pig zygote indicated a 22% increase in the pronucleus volume by DNA injection. The PCR analyses of the tail DNA of new born offspring indicated that 0.8% (4/493), 4.8% (22/463), 0.8% (3/367) and 0.9% (2/221) of the injected eggs in mice, rats, rabbits and pigs, respectively, developed into transgenic offspring. Some of the founder animals in all four species expressed the transgene in the mammary gland which was confirmed in hGH mRNA by RT-PCR and/or hGH peptide in Witch's milk with ELISA. These results suggest that the maximum volume of DNA solution injectable into the pronucleus is a possible factor explaining the species differences in the production of transgenic animals.     

Interferon expression in the testes of transgenic mice leads to sterility

A plasmid containing the mouse interferon-alpha 1 gene under control of the mouse metallothionein-I promoter was used for the construction of transgenic mice. Four transgenic mice (two males and two females) were obtained containing 1 to over 10 copies of the introduced DNA. Both males appeared to be sterile. One of the female mice founded a transgenic strain in which the foreign DNA was transmitted to her offspring in a Mendelian fashion. In this strain most male animals are sterile or turn sterile with time. Northern blot analysis of several tissues of these animals shows that expression of the introduced interferon gene occurs only in the testis. In some of the animals biologically active interferon could also be detected in testes homogenates. Histological examination of testis tissue shows an ongoing degeneration of spermatogenic cells leading to calcium deposits and complete atrophy of the seminiferous tubules.     

Unstable expression of transgene is associated with the methylation of CAG promoter in the offspring from the same litter of homozygous transgenic mice

Transgenic animals have been established for studying gene function, improving animals’ production traits, and providing organ models for the exploration of human diseases. However, the stability of inheritance and transgene expression in transgenic animals has gained extensive attention. The unstable expression of transgene through DNA methyltransferase (DNMT) targeting to the methylation of transgenic DNA such as CAG promoter and Egfp coding region in homozygous transgenic animals is still unknown. In the present study, the offspring from the same litter of homozygous transgenic mice carrying ubiquitously expressed enhanced green fluorescence protein driven by CMV early enhancer/chicken β-actin (CAG) promoter was observed to have unstable expression of transgene Egfp, quantitative PCR, western blot and bisulfite sequencing were conducted to quantify the expressional characteristics and methylation levels in various tissues. The correlation between transgene expression and methylation was analyzed. We have found that transgene expression is dependent on the methylation of CAG promoter, but not Egfp coding region. We have also characterized the correlation between the methylation of CAG promoter and DNMT, and found that only Dnmt3b expression is correlated with the methylation of CAG promoter. In conclusion, Dnmt3b-related methylation of CAG promoter can inhibit the transgene expression and may result in the unstable expression of transgene in the offspring from the same litter of homozygous transgenic mice.     

体内系统转基因新方法

建立了一种全新而高效的制备转基因动物新方法.无需外科手术,将含有绿色荧光蛋白的重组质粒直接多次多点反复注射到雄性ICR小鼠的睾丸内.几周后,上述雄性动物与自然发情的雌性交配,制备转基因动物.经PCR检测、DNA印迹,实验结果表明:F1代小鼠转基因阳性率为41%.经DNA印迹证明:外源基因已经整合到子代转基因动物的基因组内并能遗传给后代.将F1代阳性鼠与正常ICR鼠交配,产生F2代转基因鼠,F2代转基因阳性率37%.上述实验结果表明,建立的体内系统转基因方法简便、高效,适用于大规模制备转基因动物,特别适用于一些大型家畜.     

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (≥75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation ≤60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (≥80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.     

Generation of transgenic goats by pronuclear microinjection: a retrospective analysis of a commercial operation (1995–2012)

Production of transgenic founder goats involves introducing and stably integrating an engineered piece of DNA into the genome of the animal. At LFB USA, the ultimate use of these transgenic goats is for the production of recombinant human protein therapeutics in the milk of these dairy animals. The transgene or construct typically links a milk protein specific promoter sequence, the coding sequence for the gene of interest, and the necessary downstream regulatory sequences thereby directing expression of the recombinant protein in the milk during the lactation period. Over the time period indicated (1995–2012), pronuclear microinjection was used in a number of programs to insert transgenes into 18,120, 1- or 2- cell stage fertilized embryos. These embryos were transferred into 4180 synchronized recipient females with 1934 (47%) recipients becoming pregnant, 2594 offspring generated, and a 109 (4.2%) of those offspring determined to be transgenic. Even with new and improving genome editing tools now available, pronuclear microinjection is still the predominant and proven technology used in this commercial setting supporting regulatory filings and market authorizations when producing founder transgenic animals with large transgenes (> 10 kb) such as those necessary for directing monoclonal antibody production in milk.     

Pronuclear injection for the production of transgenic mice

Transgenic mice have been instrumental in dissecting the role of various neuronal proteins under both physiological and pathological conditions. Pronuclear injection is the most widely used protocol for the generation of transgenic mice. Here, we describe all steps involved from DNA purification to the set up of a mouse colony including vasectomy, injection of the DNA into a donor zygote, transfer of injected zygotes into recipient foster mice, screening of offspring and establishment of transgenic mouse lines. We discuss the use of neuron-specific promoters to express proteins with a role in Alzheimer disease. Transgenic expression of a truncated form of the microtubule-associated protein tau (delta tau) is used as an example for the anticipated results.     

利用卵胞浆精子注射（ICSI）技术生产转基因小鼠

在掌握小鼠ICSI技术的基础上,进行了ICSI技术生产转基因小鼠的研究。来自成年KM小鼠附睾尾的精子,使用未加抗冻剂的HEPES-CZB溶液,在液氮中冻融1次后,用于本实验。解冻精子与DNA混匀1min后,精子头被显微注射到B6D2F1小鼠成熟卵母细胞质中。精子头与pEGFP-N1环状DNA共注射生产的ICSI受精卵,在CZB溶液中培养至囊胚期时,39.1%(9/23)的囊胚表达GFP基因。精子注射后6h,直接移植ICSI受精卵后,7只妊娠受体一共产仔30只,效率为23.8%(30/126)。Southernblot分析其中16只小鼠发现,3只(18.8%)转基因小鼠同时整合了GFP和Neomycin基因,它们全部来自精子和线性DNA混合的实验组(阳性效率为33.3%,3/9),相反,精子与环状质粒DNA共注射生产的7只ICSI后代中,没有检测到外源基因。转基因小鼠整合的外源基因能够传递给它们后代。结果说明,利用ICSI技术可以高效地生产转基因小鼠,宿主基因组可能更容易整合线性化的外源基因。     

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.     

In vivo gene transfer to mouse spermatogenic cells using green fluorescent protein as a marker

Combination of the DNA injection into seminiferous tubules and the subsequent in vivo electroporation (EP) has become an efficient and convenient assay system for spermatogenic-specific gene expression during spermatogenesis of mice. In this study, we made methodological modifications to enhance the transfection efficiency, and evaluated the possibility of this technique to generate transgenic offspring using green fluorescent protein (GFP) as a marker. After the in vivo gene transfer, GFP expression could be monitored easily and repeatedly on the surface of the testis of live mice under fluorescent microscopy. The serial sections of the transfected testis revealed that transient expression of GFP was extended even in the innermost region of the testis uniformly, but confined to spermatogenic cells and Sertoli cells within the seminiferous tubules. Furthermore, long-lasting GFP expression could be detected in the spermatogenic cells even 2 months after EP. Natural mating with normal adult females revealed that 65% of the transfected males maintained fertilizable ability and could generate their offspring normally. Germ-line transmission of the GFP vector to the offspring was checked under fluorescent microscopy, but no transgenic offspring has been detected up to now. These results suggest that the application of additional techniques, such as cell sorting for GFP-positive germ cells followed by nuclear transfer to the oocytes, would make this method as a novel strategy for generating transgenic animals. J. Exp. Zool. 286:212-218, 2000.     

Efficient metaphase II transgenesis with different transgene archetypes.

Mammalian genome characterization and biotechnology each require the mobilization of large DNA segments to produce transgenic animals. We recently showed that mouse metaphase II (mII) oocytes could efficiently promote transgenesis (mII transgenesis) when coinjected with sperm and small (<5 kilobases) ubiquitously expressed transgenes (tgs). We have extended this work and now report that mII transgenesis can readily be applied to a range of larger tgs (11.9-170 kilobases), including bacterial and mammalian artificial chromosome (BAC and MAC) constructs. The efficiency of large-construct mII transgenesis was at least as high as that with small constructs; 11-47% of offspring carried the large tgs. More than 95% of these transgenic founders transmitted the tg to offspring. These data demonstrate the ability of mII transgenesis to deliver large tgs efficiently.     

Efficient production of transgenic mice by intracytoplasmic injection of streptolysin‐O‐treated spermatozoa

Many methods for efficient production of transgenic animals for biomedical research have been developed. Despite great improvements in transgenesis rates resulting from the use of intracytoplasmic sperm injection (ICSI), the ICSI‐based sperm‐mediated gene‐transfer (iSMGT) technique is still not optimal in terms of sperm permeabilization efficiency and subsequent development. Here, we demonstrate that streptolysin‐O (SLO) can efficiently permeabilize mouse spermatozoa, leading to improved developmental competence and high transgenesis rates in iSMGT embryos and pups. In particular, the most efficient production of iSMGT‐transgenic embryos resulted from pretreatment with 5 U/ml SLO for 30 min and co‐incubation with 1.0 ng/µl of an EGFP expression vector. By incubating spermatozoa with Cy‐3‐labelled DNA, we found that fluorescence intensity was prominently detected in the head region of SLO‐treated spermatozoa. In addition, blastocyst development rate and blastomere survival were greatly improved by iSMGT using SLO‐treated spermatozoa (iSMGT‐SLO) as compared to freeze‐thawed spermatozoa. Consistent with this, a high proportion of transgenic offspring was obtained by iSMGT‐SLO after transfer into foster mothers, reaching 10.6% of the number of oocytes used (42.3% among pups). Together with successful germline transmission of transgenes in all founders analyzed, our data strongly suggest that SLO makes spermatozoa amenable to exogenous DNA uptake, and that the iSMGT‐SLO technique is an efficient method for production of transgenic animals for biomedical research. Mol. Reprod. Dev. 80: 233–241, 2013. © 2013 Wiley Periodicals, Inc.     

A Rapid and Simple PCR-based Method for Analysis of Transgenic Fish using a Restricted Amount of Fin Tissue

The protocol described in this paper offers a simple and rapid method for PCR analysis of transgenes using a restricted amount of fin tissue from small-sized transgenic fish. A simple preparation of fin lysate using a buffer containing a low concentration of an ionic detergent, SDS (0.01%), followed by neutralization with a second buffer containing higher concentrations of non-ionic detergents NP40 (2%) and Tween 20 (2%) consistently provides a reliable quantity of high-quality DNA template for PCR amplification of transgenes. Based on this protocol, transgenic fish can be clearly distinguished from non-transgenic fish using PCR in a rapid and reproducible manner. Tedious DNA purifications are avoided while fidelity of amplification and efficient identification of transgenic fish are maintained.     

Polymerase chain reaction analysis of transgenic plants contaminated byAgrobacterium

Agrobacterium-mediated genetic transformation is a method of choice for the development of transgenic plants. The presence of latentAgrobacterium that multiplies in the plant tissue in spite of antibiotic application confounds the results obtained by polymerase chain reaction (PCR) analysis of putative transgenic plants. The presence ofAgrobacterium can be confirmed by amplification of eitherAgrobacterium chromosomal genes or genes present out of transfer DNA (T-DNA) in the binary vector. However, the transgenic nature ofAgrobacterium-contaminated transgenic plants cannot be confirmed by PCR. Here we report a simple protocol for PCR analysis ofAgrobacterium-contaminated transgenic plants. This protocol is based on denaturation and renaturation of DNA. The contaminating plasmid vector becomes double-stranded after renaturation and is cut by a restriction enzyme having site(s) within the PCR amplicon. As a result, amplification by PCR is not possible. The genomic DNA with a few copies of the transgene remains single-stranded and unaffected by the restriction enzyme, leading to amplification by PCR. This protocol has been successfully tested with 4 different binary vectors and 3Agrobacterium tumefaciens strains: EHA105, LBA4404, and GV3101.     

Production of transgenic mice expressing the goat H-FABP gene by intratesticular injection

The objective of this study was to explore the possibility of obtaining stable transgenic animals by intratesticular injection. The recombinant vector pEGFP-H-FABP expressing the goat heart-type fatty acid binding protein and green fluorescent protein was mixed with liposome complexes and randomly injected into the testes of mice. Testicular section, fluorescence, and DNA detection assays of mouse sperm were performed to determine the integration of foreign DNA. The results showed that foreign DNA was successfully expressed in the treated mice. Furthermore, the expression and function of the foreign gene were analyzed in F₁ generation and F₂ generation mice at different levels, with the positive rates of foreign gene transfer into the F₁ and F₂ generations being 4.0 and 30.23 %, respectively. These results strongly support testicular injection as an effective method of producing transgenic animals and indicate that foreign genes can be stably passed on to the offspring. This research has theoretical and practical implications for the improvement in the quality of laboratory animals and for gene therapy.     

Techniques of embryo transfer and facility decontamination used to improve the health and welfare of transgenic mice

'Reduction' and 'Refinement' can be achieved in transgenic mouse studies by re-deriving transgenic mouse lines and subsequently maintaining them under high standards of husbandry in a unit with restricted access. This report describes the initial steps of a project to improve the health and welfare of transgenic mice at the European Molecular Biology Laboratory (EMBL), by re-deriving transgenic lines as microbiologically defined animals to be maintained in a barrier unit in a newly constructed animal facility. A pilot study showed that it was possible to transfer embryos obtained from contaminated donor mice in the old facility to specific pathogen free recipients housed in a ventilated cabinet in the new unit, without concomitant carry over of disease. The offspring born following embryo transfer were of high health status and did not show any evidence of contamination with any of the pathogens present in the mice in the old animal unit. Antibodies to various murine viruses (mouse hepatitis virus (MHV), rota virus, reo-3 virus, Theilers encephalomyelitis virus, adenovirus) and parasites were present in sentinel animals from the old animal house whereas the re-derived animals were found to be free of virus antibodies and parasites. Therefore the methods used were considered to be successful in terms of disease prevention and enhancement of welfare. The barrier unit was sterilized without the use of formaldehyde or related substances, to minimize the risks to personnel and to the environment from using potentially dangerous substances. From the results of in vitro and in vivo screening, the protocol for sterilization described here was found to be effective in achieving microbiological sterility of the barrier unit and was cost effective.