Efficient generation of transgenic mice with intact yeast artificial chromosomes by intracytoplasmic sperm injection

The production of animals with large transgenes is an increasingly valuable tool in biotechnology and for genetic studies, including the characterization and manipulation of large genes and polygenic traits. In the present study, we describe an intracytoplasmic sperm injection (ICSI) method for the stable incorporation and phenotypic expression of large yeast artificial chromosomes (YAC) constructs of submegabase and megabase magnitude. By coinjecting spermatozoa and YACs into metaphase II oocytes, we were able to produce founders exhibiting germline transmission of an intact and functional transgene of 250 kilobases, carrying the mouse tyrosinase locus, used here as a reporter gene to rescue the albinism of recipient mice. More than 35% transgenesis was obtained for this YAC transgene. When compared with the pronuclear microinjection standard method, the efficiency of the ICSI-mediated YAC transfer system was significantly greater. In summary, we describe, for the first time, stable incorporation in the host genome and correct phenotypic expression of large DNA constructs mediated by ICSI.     

Size Matters: Use of YACs,BACs and PACs in Transgenic Animals

In 1993, several groups, working independently, reported the successful generation of transgenic mice with yeast artificial chromosomes (YACs) using standard techniques. The transfer of these large fragments of cloned genomic DNA correlated with optimal expression levels of the transgenes, irrespective of their location in the host genome. Thereafter, other groups confirmed the advantages of YAC transgenesis and position-independent and copy number-dependent transgene expression were demonstrated in most cases. The transfer of YACs to the germ line of mice has become popular in many transgenic facilities to guarantee faithful expression of transgenes. This technique was rapidly exported to livestock and soon transgenic rabbits, pigs and other mammals were produced with YACs. Transgenic animals were also produced with bacterial or P1-derived artificial chromosomes (BACs/PACs) with similar success. The use of YACs, BACs and PACs in transgenesis has allowed the discovery of new genes by complementation of mutations, the identification of key regulatory sequences within genomic loci that are crucial for the proper expression of genes and the design of improved animal models of human genetic diseases. Transgenesis with artificial chromosomes has proven useful in a variety of biological, medical and biotechnological applications and is considered a major breakthrough in the generation of transgenic animals. In this report, we will review the recent history of YAC/BAC/PAC-transgenic animals indicating their benefits and the potential problems associated with them. In this new era of genomics, the generation and analysis of transgenic animals carrying artificial chromosome-type transgenes will be fundamental to functionally identify and understand the role of new genes, included within large pieces of genomes, by direct complementation of mutations or by observation of their phenotypic consequences.     

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.     

Preparation of intact yeast artificial chromosome DNA for transgenesis of mice

Transgenesis with large DNA molecules such as yeast artificial chromosomes (YACs) has an advantage over smaller constructs in that an entire locus and all its flanking cis-regulatory elements are included. The key to obtaining animals bearing full-length transgenes is to avoid physical shearing of the DNA during purification and microinjection. This protocol details how to prepare intact YAC DNA for transgenesis of mice and involves separation of YAC DNA from yeast chromosomal DNA by pulsed field gel electrophoresis, concentration to a range suitable for microinjection by second dimension electrophoresis and enzymatic digestion of matrix-embedded YAC DNA to produce a solution that can be injected. The YAC is maintained in an agarose gel matrix to avoid damage until the final steps before microinjection. Special precautions are also taken during the microinjection protocol. Transgenesis efficiency is approximately 15%; most animals carry 1-5 copies of the desired locus. This method takes 6 d for completion.     

转基因动物乳腺生物反应器的发展概况及人工染色体在其中的应用

转基因动物乳腺生物反应器位点效应的影响是制备转基因动物乳腺生物反应器过程中的主要问题。酵母人工染色体（YAC）和细菌人工染色体（BAC）具有容量大的特性,可以将乳蛋白的整个基因座包括所有调控序列全部装载进去,有可能克服位点效应的影响,是一种理想的载体。YAC和BAC载体转基因技术可能成为避开基因打靶获得高效表达的转基因动物乳腺生物反应器的另一途径.     

Transgenic mice carrying yeast artificial chromosomes

The generation of transgenic mice with yeast artificial chromosomes (YACs) has proven to be a valuable system to: (1) study gene structure-function relationships; (2) produce mouse models of human disease; (3) complement mouse mutants; (4) generate mice bioreactors; and (5) screen YAC libraries in vivo. Continued refinement of current techniques and development of new protocols should encourage widespread adaptation of this strategy for these and other applications. Use of whole loci as transgenes is an important improvement in murine transgenesis because it results in a more realistic pattern and level of gene expression during ontogeny. Application of this technology to develop human artificial chromosomes (HACs) might provide the next generation of gene therapy vectors that will overcome most of the problems and barriers associated with current vector systems.     

YAC transgenesis in farm animals: Rescue of albinism in rabbits

The generation of transgenic mice with mammalian genes cloned in yeast artificial chromosomes (YACs) has generated great interest in the field of gene transfer into livestock. Many of the problems associated with standard transgenesis—such as lack of crucial regulator elements and position effects related to the integration site, which lead to variation in expression levels irrespective of the dose of the transgene—have been practically overcome. The large size of YAC-derived gene constructs (in excess of 1 Mb) facilitates the presence and transfer of all elements required for the faithful regulation of a gene. With the experiments discussed in this report, we have addressed the possibility of applying the obvious advantages of YAC transgenesis to farm animals. We have generated transgenic rabbits carrying a 250 kb YAC covering the mouse tyrosinase gene by pronuclear microinjection, and thus rescued the albino phenotype of the transgenic individuals. To date, this is the first demonstration of a successful transfer of large genetic units into the germ line of farm animals. This development might improve the occurrence of transgene expression at physiological levels and specific sites in livestock. YAC transgenesis therefore will be applied in genetic engineering, for example, in the production of pharmacologically interesting proteins encoded by large gene units and generating transgenic donors for xenotransplantation. © 1996 Wiley-Liss, Inc.     

Effect of transgene concentration, flanking matrix attachment regions, and RecA-coating on the efficiency of mouse transgenesis mediated by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) of DNA-loaded sperm cells has been shown to be a valuable tool for the production of transgenic animals, especially when DNA constructs with submegabase magnitude are used. In order to optimize and to understand the mechanism of the ICSI-mediated transgenesis, we have evaluated the impact of transgene DNA concentration, transgene flanking with nuclear matrix attachment regions (MARs), and the use of recombinase A (RecA)-coated DNA on the efficiency of mouse transgenesis production by ICSI. Presented data include assays with three DNA constructs; an enhanced green fluorescent protein (EGFP) plasmid of 5.4 kb, this plasmid flanked with two MAR elements (2.3 Kb of the human beta-interferon domain boundaries), and a yeast artificial chromosome (YAC) construct of ~510 kb (the largest transgenic construct introduced by ICSI that we have seen reported). ICSI-mediated transgenesis was done in the B6D2 mouse strain using different concentrations for each construct. Analysis of generated data indicated that ICSI allows the use of higher DNA concentrations than the ones used for pronuclear microinjection, however, when a certain threshold is exceeded, embryo/fetal viability decrease dramatically. In addition, independently of the transgene concentration tested, transgene flanking with MAR sequences did not have a significant impact on the efficiency of this transgenesis method. Finally, we observed that although the overall efficiency of ICSI-mediated transgenesis with fresh spermatozoa and RecA-complexed DNA was similar to the one obtained with the common ICSI-mediated transgenesis approach with frozen-thawed spermatozoa and RecA free DNA, this method was not as efficient in maintaining a low frequency of founder animal mosaicism, suggesting that different mechanisms of transgene integration might result from each procedure.     

Generation of transgenic rats with YACs and BACs: preparation procedures and integrity of microinjected DNA.

The aim of the present study was to investigate differences in the methods for preparing a large DNA fragment to be used for making transgenic rats from the standpoint of transgenic production efficiency and integrity of the introduced gene. In yeast artificial chromosome (YAC) transgenesis, three methods for preparing DNA for microinjection were compared: amplification of YAC in yeast (AMP), amplification of YAC in yeast and removal of the amplification element (AMP/RE), and no amplification of the YAC in yeast (AMP-). Production efficiency per microinjected ovum with DNA by the AMP method was four times higher than that by the AMP/RE and AMP-. Based on these results, we favor the AMP method in spite of the thymidine kinase gene-induced male sterility. In bacterial artificial chromosome (BAC) transgenesis, linear DNA fragments for microinjection prepared by three kinds of purification procedures were compared: Not I digestion and CsCl gradient ultra-centrifugation (Prep. 1), CsCl gradient ultra-centrifugation, Not I digestion, gel electrophoresis, and beta-agarase digestion (Prep. 2), and CsCl gradient ultra-centrifugation, Not I digestion, pulse field gel electrophoresis, and beta-agarase digestion (Prep. 3). Although the efficiency of producing transgenic rats was similar with all these three DNA preparations, integration of the intact DNA fragment only occurred with the Prep. 3 procedure. We therefore favor the Prep. 3 method for preparing BAC DNA fragments. These results indicate that the method used to prepare a large DNA fragment such as YAC and BAC DNAs is important in order to produce transgenic rats with an intact transgene.     

Use of frozen-thawed oocytes for efficient production of normal offspring from cryopreserved mouse spermatozoa showing low fertility

Freezing of spermatozoa and unfertilized oocytes is a useful tool for the conservation of mouse genetic resources. However, the proportion of frozen-thawed oocytes fertilized with spermatozoa in vitro is low because spermatozoa, especially those frozen-thawed, can not penetrate into oocytes because of hardening of the zona pellucida following premature release of cortical granules. To produce offspring efficiently from cryopreserved transgenic mouse gametes, we fertilized frozen-thawed gametes by using intracytoplasmic sperm injection (ICSI) and assessed pre- and postimplantation development of embryos. Compared with fresh unfertilized oocytes, frozen-thawed unfertilized oocytes were highly tolerant to damage by injection, as the survival rates after injection of frozen spermatozoa were 51 and 78%, respectively. Frozen-thawed oocytes that survived after sperm injection developed normally to the blastocyst stage and gave rise to offspring. Moreover, offspring with transgenes also were obtained from frozen gametes fertilized by ICSI. These results demonstrate that ICSI is an efficient technique for producing offspring from transgenic spermatozoa showing low fertility and that use of frozen-thawed oocytes leads to conservation of genetic resources because suboptimally preserved gametes are not wasted.     

High-resolution structural analysis of biolistic transgene integration into the genome of wheat

Transformation of plant genomes by biolistic methods has become routine over the past decade. However, relatively little is known about how transgenes are physically integrated into the host genome. Using a high-resolution physical mapping technique, fluorescence in situ hybridization on extended DNA fibers (fiber-FISH), 13 independent transgenic wheat lines were analyzed to determine the structural arrangement of stably inherited transgenes in host-plant chromosomes. Twelve transgenic lines were transformed with a single plasmid and one line was co-transformed with two separate plasmids, which co-segregated genetically. Three basic integration patterns were observed from the fiber-FISH experiments: Type I, large tandemly repeated integration; Type II, large tandem integrations interspersed with unknown DNA; and Type III, small insertions, possibly interspersed with unknown DNA. Metaphase FISH showed that the integration of transgenes was in both hetero- and euchromatic, as well as proximal, interstitial and distal, regions of the chromosomes. In the transgenic plants, the type of promotor used, rather than the chromosomal site of transgene integration, was most critical for transgene expression. The integration of the transgenes was not associated with detectable chromosomal rearrangements. Received: 25 August 2000 / Accepted: 31 October 2000     

Generation of transgenic cattle by lentiviral gene transfer into oocytes

The potential benefits of transgenic cattle range from the production of large quantities of pharmaceutically relevant proteins to agricultural improvement. However, the production of transgenic cattle is presently time-consuming and expensive because of the inefficiency of the classical DNA microinjection technique. Here, we report the use of lentiviruses for the efficient generation of transgenic cattle. Initial attempts to produce transgenic cattle by lentiviral infection of preimplantation embryos were not successful. In contrast, infection of bovine oocytes with lentiviral vectors carrying an enhanced green fluorescent protein (eGFP) expression cassette followed by in vitro fertilization resulted in the birth of transgenic calves. Furthermore, all of the calves generated by infection of oocytes were transgenic, and 100% of these animals expressed eGFP as detected by in vivo imaging and Western blotting. In addition, a transgenic calf was produced by infection of fetal fibroblasts followed by nuclear transfer into enucleated oocytes. Taken together, after adjusting lentiviral transgenesis to cattle, unprecedented high transgenesis and expression rates were achieved.     

The creation of transgenic pigs expressing human proteins using BAC-derived, full-length genes and intracytoplasmic sperm injection-mediated gene transfer

In most transgenic (Tg) animals created to date, a transgene consisting of the minimum promoter region linked to a cDNA has been used. However, transgenes on small plasmids are susceptible to the position effect, increasing the difficulty of controlling transgene expression. In this study, we attempted to create Tg pigs by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) using two large genomic transgenes derived from a bacterial artificial chromosome (BAC) containing the full genomic region encoding two human proteins, type I collagen and albumin. The production efficiencies (Tg piglets/live offspring) of type I collagen and albumin Tg pigs were 11.8% (6/51) and 18.2% (2/11), respectively. In all of the Tg pigs examined by real-time PCR analysis, tissue-specific expression of the transgene was confirmed (type I collagen: skin, tendon, vessels, genitalia; albumin: liver). The production of human proteins derived from BAC transgenes was also confirmed. Fluorescence in situ hybridization analysis indicated that the BAC transgenes transferred into porcine oocytes by ICSI-MGT were integrated into single or multiple sites on the host chromosomes. These data demonstrate that Tg pigs expressing human proteins in a tissue-specific manner can be created using a BAC transgenic construct and the ICSI-MGT method.     

A new method for generating point mutations in bacterial artificial chromosomes by homologous recombination in Escherichia coli

Bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs), which contain large fragments of genomic DNA, have been successfully used as transgenes to create mouse models of dose-dependent diseases. They are also potentially valuable as transgenes for dominant diseases given that point mutations and/or small rearrangements can be accurately introduced. Here, we describe a new method to introduce small alterations in BACs, which results in the generation of point mutations with high frequency. The method involves homologous recombination between the original BAC and a shuttle vector providing the mutation. Each recombination step is monitored using positive and negative selection markers, which are the Kanamycin-resistance gene, the sacB gene and temperature-sensitive replication, all conferred by the shuttle plasmid. We have used this method to introduce four different point mutations and the insertion of the β-galactosidase gene in a BAC, which has subsequently been used for transgenic animal production.     

Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes

Transgenic mice are widely used in biomedical research to study gene expression, developmental biology, and gene therapy models. Bacterial artificial chromosome (BAC) transgenes direct gene expression at physiological levels with the same developmental timing and expression patterns as endogenous genes in transgenic animal models. We generated 707 transgenic founders from 86 BAC transgenes purified by three different methods. Transgenesis efficiency was the same for all BAC DNA purification methods. Polyamine microinjection buffer was essential for successful integration of intact BAC transgenes. There was no correlation between BAC size and transgenic rate, birth rate, or transgenic efficiency. A narrow DNA concentration range generated the best transgenic efficiency. High DNA concentrations reduced birth rates while very low concentrations resulted in higher birth rates and lower transgenic efficiency. Founders with complete BAC integrations were observed in all 47 BACs for which multiple markers were tested. Additional founders with BAC fragment integrations were observed for 65% of these BACs. Expression data was available for 79 BAC transgenes and expression was observed in transgenic founders from 63 BACs (80%). Consistent and reproducible success in BAC transgenesis required the combination of careful DNA purification, the use of polyamine buffer, and sensitive genotyping assays.     

Factors affecting production of transgenic rats by ICSI-mediated DNA transfer: effects of sonication and freeze-thawing of spermatozoa, rat strains for sperm and oocyte donors, and different constructs of exogenous DNA

Factors affecting the efficiency of producing transgenic rats by intracytoplasmic sperm injection (ICSI)-mediated DNA transfer were investigated. Epididymal spermatozoa from Sprague-Dawley (SD) rats were sonicated and/or frozen-thawed for cutting the tail and membrane disruption. The sperm heads were exposed for 1 min to different concentrations (0.02-2.5 microg/ml) of 3.0 kb enhanced green fluorescent protein (EGFP) DNA solution, and then microinjected into the denuded F1 hybrid (Donryu x LEW) rat oocytes. The optimal concentration of EGFP DNA solution was 0.1 microg/ml, as determined by the in vitro developmental competence into morulae/blastocysts of the ICSI oocytes and the EGFP expression of the resultant embryos. The efficiency of producing transgenic rat offspring (per transferred zygote) was 2.8%, 1.6%, and 3.3% in the oocytes injected with sonicated, frozen-thawed, and sonicated + frozen-thawed sperm heads, respectively. The founder transgenic rats carrying the EGFP gene transmitted their transgenes to their progeny according to the Mendelian fashion, suggesting the stable incorporation of the transgenes into the rat genomes. Four rat strains (F344, LEW, Donryu, and SD) were compared for their suitability as sperm/oocyte donors for the production of transgenic rats by ICSI with sonicated, frozen-thawed and solution of EGFP DNA-exposed sperm heads. The efficiency of producing transgenic rats in the SD strain (8.2%) was higher than that in the LEW strain (0.9%), while those in the F344 and Donryu strains (4.3%-4.4%) were intermediate. One plasmid DNA (Fyn, 5.0 kb) and two BAC DNA (BAC/Fyn, 208 kb; Svet1/IRES-Cre, 186 kb) were successfully introduced into the SD rat genomes via ICSI, with the producing efficiencies of 2.8%, 0.9%, and 2.4%, respectively.     

A novel transgenic mice model for venous malformation

Vascular anomalies are most commonly seen in the head and neck region, and there is no animal model available of this disease until now. The purpose of this study was to construct a conditional murine polyomavirus middle T antigen gene (PyMT) transgenic mice model, in order to provide a basis for the treatment of vascular anomalies in vivo, as well as the study of PyMT's molecular function. A new conditional transgenic vector based on Tet-On system was constructed successfully. After the experiment in vitro, pronuclear microinjection method was used to introduce the purified transgene into the chromosomes of fertilized mice eggs, and five transgenic positive mice were obtained. The transgenic positive animals went down to future generation by hybridization. After induction of PyMT's expression in the F1 generation, three transgenic mice developed venous malformation which was confirmed histopathologically. The mice model generated could be used as a tool to study venous malformation, as well as the function of PyMT gene.     

Establishment of an efficient BAC transgenesis protocol and its application to functional characterization of the mouse Brachyury locus.

Transgenesis using large DNA such as YAC or BAC has extended the range of applications in functional genomics. Here we describe an efficient BAC transgenesis protocol using a simple BAC DNA preparation method adopted from YAC DNA purification methods. This method allowed us to isolate BAC DNA from small scale culture of BAC-containing cells in sufficient quantity and purity for microinjection. More than 40 founders have been produced with linearized BAC DNA prepared by this method, and 85% of them contained intact BAC transgenes. In contrast, when circular BAC DNA was injected, an approximately three-fold reduction of transgene integration rate was observed and fewer intact transgene integrations were obtained. A line of transgenic mice carrying a 170-kb BAC clone generated in this way successfully rescued tail and embryonic lethality phenotypes of the mouse Brachyury (T) mutants, further demonstrating the utility of this method in functional analysis of the mouse genome.     

High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes

Whereas bacterial artificial chromosomes (BACs) offer many advantages in studies of gene and protein function, generation of seamless, precisely mutated BACs has been difficult. Here we describe a counterselection-based recombineering method and its accompanying reagents. After identifying intramolecular recombination as the major problem in counterselection, we built a strategy to reduce these unwanted events by expressing Redβ alone at the crucial step. We enhanced this method by using phosphothioated oligonucleotides, using a sequence-altered rpsL counterselection gene and developing online software for oligonucleotide design. We illustrated this method by generating transgenic mammalian cell lines carrying small interfering RNA-resistant and point-mutated BAC transgenes. Using this approach, we generated mutated TACC3 transgenes to identify phosphorylation-specific spindle defects after knockdown of endogenous TACC3 expression. Our results highlight the complementary use of precisely mutated BAC transgenes and RNA interference in the study of cell biology at physiological expression levels and regulation.