Effects of cryopreservation of pronuclear-stage rabbit zygotes on the morphological survival, blastocyst formation, and full-term development after DNA microinjection

The objectives of this study were to examine the freezing sensitivity of pronuclear-stage rabbit zygotes and to produce transgenic rabbits using the cryopreserved zygotes. Zygotes were cryopreserved either by one of two vitrification protocols or by one of the two conventional freezing protocols. The morphological survival rates of zygotes subjected to two-step freezing in 1.5 M ethylene glycol and 0.1 M sucrose (74%) or to vitrification in 7.2 M ethylene glycol and 1.0 M sucrose (81%) were higher than those subjected to freezing in 1.5 M DMSO (46%) or to vitrification in a mixture of 2.0 M DMSO, 1.0 M acetamide, and 3.0 M propylene glycol (41%). But the in vitro development into blastocysts of zygotes cryopreserved by vitrification (17%) or to a lesser extent by freezing (52%) was impaired, when compared to that of fresh control zygotes (89%). Next, a fusion gene composed from bovine aS1-casein promoter and a human GH structural gene (2.8 kb) was microinjected into the pronucleus of rabbit zygotes frozen-thawed in ethylene glycol and sucrose. Then, the presence of exogenous DNA in the genome of newborn offspring was determined by PCR. The post-injection survival of frozen zygotes (97%) was the same as that of fresh control zygotes (96%). However, of 18 offspring derived from 414 frozen-thawed and DNA-injected zygotes, no transgenic rabbits were produced. Of 52 offspring derived from 403 DNA-injected fresh zygotes, 3 transgenic rabbits were found. Here we report the first rabbit offspring resulting from zygotes cryopreserved at the pronuclear-stage, although the cryopreservation procedure employed must be improved if zygotes are to be used for systematic production of transgenic rabbits.     

绿色荧光蛋白基因在转基因小鼠体内的表达

建立绿色荧光蛋白（GFP）转基因小鼠,继而传代建系。采用显微注射法,将GFP基因注入FVB／NJ小鼠受精卵原核内,获得子代鼠。分娩后3周剪取仔鼠尾,提取基因组DNA,应用PCR、Southern印迹技术进行整合检测。结共用雌性小鼠200只,注射受精卵1586枚,移植卵数386枚,受体鼠32只,怀孕鼠4只,子代鼠18只,有4只为阳性：取2只首建鼠的胚胎,在荧光显微镜下观察GFP表达明显,表明初步获得了转绿色荧光蛋白基因小鼠,     

The effect of the micromanipulations used for transgenosis on mouse development]

Non-specific effects of micromanipulation techniques used for producing transgenic mice on processes of embryonic development were studied. Zygotes obtained from C57BL and BALBxDD mice were treated as follows: (1) incubated in culture medium; (2) the male pronucleus punctured with a glass microneedle; (3) microinjected with a buffer solution; and (4) DNA (mouse P-35 oncogene with human insulin gene promoter) injected into the male pronucleus. Then zygotes were transferred into oviducts of syngeneic or allogeneic pseudopregnant females. Such treatment resulted in the intrauterine death of embryos, as well as in birth of the dead or non-viable offspring with numerous defects of development. Zygote pronucleus puncturing is the most damaging manipulation, since its effect exceeds that of the zygote incubation and is comparable with the effect of buffer of DNA injections.     

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.     

SHORT COMMUNICATION: Double pronuclei injection of DNA into zygotes increases yields of transgenic mouse lines

Transgenic mice are increasingly used for gene function and regulation studies of mammalian genes. A major limitation is the necessity to produce a large number of founder animals to obtain one line with the desired expression pattern. We developed a method, the 'double pronuclei injection', that doubles the yield of transgenic mouse lines obtained from each injection session, thereby reducing the time, effort and costs of generating transgenic mice. Three transgenic vectors were microinjected into the male and female pronuclei of zygotes. Approximately half of the resulting born mice were transgenic. This represented a 60% increase in the yield of founders per injected zygote, and a 100% increase in the yield of transgenic mice per born animal, when compared to yields obtained using single pronucleus injection. This method should prove useful for generating large numbers of transgenic mice for gene regulation studies and for conditional gene ablation     

Production of transgenic miniature pigs by pronuclear microinjection

Miniature pig is an attractive animal for a wide range of research fields, such as medicine and pharmacology, because of its small size, the possibility of breeding it under minimum environmental controls and the physiology that is potentially similar to that of human. Although transgenic technology is useful for the analysis of gene function and for the development of model animals for various diseases, there have not yet been any reports on producing transgenic miniature pig. This study is the first successful report concerning the production of transgenic miniature pig by pronuclear microinjection. The huntingtin gene cloned from miniature pig, which is a homologue of candidate gene for Huntington's disease, connected with rat neuron-specific enolase promoter region, was injected into a pronucleus of fertilized eggs with micromanipulator. The eggs were transferred into the oviduct of recipient miniature pigs, whose estrus cycles were previously synchronized with a progesterone analogue. A total of 402 injected eggs from 171 donors were transferred to 23 synchronized recipients. Sixteen of them maintained pregnancy and delivered 65 young, and one resulted in abortion. Five of the 68 offspring (three of which were aborted) were determined to have transgene by PCR and Southern analysis. The overall rate of transgenic production was 1.24% (transgenic/injected eggs). This study provides the first success and useful information regarding production of transgenic miniature pig for biomedical research.     

Comparison between PMSG- and FSH-induced superovulation for the generation of transgenic rats

Superovulation protocols using single injections of pregnant mare's serum gonadotropin (PMSG) or minipumps with follicle-stimulating hormone (FSH) were compared in immature Sprague-Dawley (SD) rats. We used the following criteria: total number of ova, rate of fertilization, in vitro embryo development, sensitivity of zygotes to the microinjection of foreign DNA into the pronucleus, and their in-vivo development after transplantation into the oviduct of a recipient. Female SD rats were stimulated with 15 IU PMSG or 10 mg FSH followed by the injection of human chorionic gonadotropin (hCG) at doses of 20 and 30 IU per female. After hCG administration, they were mated with males of the same strain and sacrificed on day 1 of pregnancy. The percentage of mated animals and the fertilization rate was similar in all groups. In rats given PMSG, the number of ovulated zygotes was hCG dose-dependent. In contrast, the dose of hCG did not influence the efficiency of superovulation in rats given FSH, which was equal to PMSG-treated rats at the optimal dose of hCG. The rates of in vitro blastocyst development (31.4 and 23.3%) and the resistance to microinjection into the pronucleus did also not differ significantly between zygotes of both studied groups. The proportion of offspring developing from microinjected zygotes after oviduct transfer (26.2 and 26.8%, respectively) and the rate of transgene integration per newborns (7.3 and 4.9%, respectively) was similar in both experimental groups. The results of this study demonstrate that superovulation of immature SD rats by PMSG is equally effective as FSH treatment and, thus, preferable for transgenic rat technology due to the lower costs and easier handling.     

激肽释放酶转基因大鼠模型的建立

目的建立KLK1转基因大鼠模型。方法腹腔注射PMSG-HCG（150-150IU/kg）超排不同周龄（4-8周）SD大鼠比较超排效果的差异,以可用于注射胚胎数作为评判标准确定最佳超排周龄。构建pBC-klk1转基因构件,经酶切、纯化后通过显微注射方法导入SD大鼠受精卵原核并移植到同期受孕的SD受体母鼠输卵管内。出生后仔鼠用PCR和Southern方法检测鼠尾DNA鉴定基因型,通过RT-PCR和免疫组化方法检测klk1基因表达。结果4-8周SD大鼠超排后分别获得受精卵14±14.8、30±15.2、13.3±13.7、13±14.7、8±5.7,5周龄大鼠超排效果最好,与其他周龄大鼠相比有显差异,P〈0.05;显微注射1538枚卵,移植685（44.53%）枚卵于31只受体输卵管中,12（12/31,38.7%）只怀孕,共产仔62只,经PCR检测获得6只阳性鼠,Southern检测3只阳性。对Southern检测阳性转基因大鼠子代进行RT-PCR检测和免疫组化分析证明klk1基因在肾脏、胰腺和乳腺内表达。结论成功建立klk1基因表达的转基因大鼠模型,该模型是高血压病研究的理想动物模型。     

DNA methylation state is preserved in the sperm-derived pronucleus of the pig zygote

DNA methylation reprogramming (DMR) during preimplantation development erases differentiation-associated, unessential epigenetic information accumulated during gametogenesis, and ultimately brings pluripotency to the resulting embryo. Two patterns of DMR of sperm-derived pronucleus have been reported in mammals. In the first, the male pronucleus is actively demethylated whereas in the second, the methylation state seems to be maintained. The maintenance-type DMR has been seen only through immunocytochemical observations, and waits to be proven by additional molecular-level evidence. We demonstrate that, in pig, paternally derived DNA methylation is preserved during pronucleus development, based on the following observations. First, immunostaining of pig zygotes at different time points showed the DNA methylation state to be balanced between parental pronuclei throughout pronucleus development. Second, bisulfite analysis of PRE-1 repetitive sequences found mono- and polyspermic eggs to have similar methylation states. Third, the methylation state of a human erythropoietin gene delivered by transgenic pig spermatozoa was maintained in the male pronucleus. Finally, 5-aza-2'-deoxycytidine treatment, which blocks re-methylation, did not show the male pronucleus to be stalled in a demethylated state. In pig zygotes, paternally derived cytosine methylation was preserved throughout pronucleus development. These findings from multilateral DMR analyses provide further support to the view that DMR occurs in a non-conserved manner during early mammalian development.     

DNA preparation method can influence outcome of transgenic animal experiments

In our continuing quest to improve the efficiency of producing transgenic animals, we have compared the influence of two transgene purification techniques on the efficiency of creating transgenic sheep and mice. Three hundred eighty-seven sheep zygotes and 2,737 mouse zygotes were microinjected with one of four transgenes. Transgenes were isolated from plasmid sequences either by agarose gel electrophoresis followed by gel extraction or by a single step sodium chloride gradient fractionation technique. Four transgenic sheep and 61 transgenic mice were produced. Both sheep and mice embryos responded similarly to transgene preparation methods. Overall, pregnancy rate was higher for recipients that received embryos injected with NaCl purified DNA (mean +/- SEM: 64 +/- 7% vs. 38 +/- 7%). Furthermore, offspring per zygote transferred (NaCl, 22 +/- 3% vs. Gel, 12 +/- 3%) and transgenics born per zygote transferred (NaCl, 3.9 +/- 0.6% vs. Gel, 1.5 +/- 0.6%) were higher when the NaCl purified DNA was used. However, the proportion of offspring born that were identified as transgenic did not differ between transgene purification methods. Transgenes responded differently to methods of preparation. One of the four genes yielded a significantly higher proportion of transgenics when the transgene was prepared by NaCl purification. These data suggest that on average the NaCl gradient purification technique results in a higher embryo survival rate to term for both sheep and mice, but the technique has no influence on rate of transgene integration.     

DNA preparation method can influence outcome of transgenic animal experiments

Abstract

In our continuing quest to improve the efficiency of producing transgenic animals, we have compared the influence of two transgene purification techniques on the efficiency of creating transgenic sheep and mice. Three hundred eighty‐seven sheep zygotes and 2,737 mouse zygotes were microinjected with one of four transgenes. Transgenes were isolated from plasmid sequences either by agarose gel electrophoresis followed by gel extraction or by a single step sodium chloride gradient fractionation technique. Four transgenic sheep and 61 transgenic mice were produced. Both sheep and mice embryos responded similarly to transgene preparation methods. Overall, pregnancy rate was higher for recipients that received embryos injected with NaCl purified DNA (mean + SEM: 64 ± 7% vs. 38 ± 7%). Furthermore, offspring per zygote transferred (NaCl, 22 + 3% vs. Gel, 12 + 3%) and transgenics born per zygote transferred (NaCl, 3.9 ± 0.6% vs. Gel, 1.5 ± 0.6%) were higher when the NaCl purified DNA was used. However, the proportion of offspring born that were identified as transgenic did not differ between transgene purification methods. Transgenes responded differently to methods of preparation. One of the four genes yielded a significantly higher proportion of transgenics when the transgene was prepared by NaCl purification. These data suggest that on average the NaCl gradient purification technique results in a higher embryo survival rate to term for both sheep and mice, but the technique has no influence on rate of transgene integration.     

An analysis of factors affecting efficiency in obtaining transgenic mice

Microinjection of exogenous DNA into male pronucleus at early stages of its formation leads to greater yield of transgenic mice than DNA introduction into male pronucleus at later stages. Dulbecco medium is more suitable for manipulation with murine zygotes than HEPES-containing Whitten medium. On the other side, albumin-free Whitten medium is better for dissolving exogenous DNA for introduction into murine zygote pronucleus as compared to Dulbecco medium and TE buffer.     

Production of human apolipoprotein(a) transgenic NIBS miniature pigs by somatic cell nuclear transfer

Most cases of ischemic heart disease and stroke occur as a result of atherosclerosis. The purpose of this study was to produce a new Nippon Institute for Biological Science (NIBS) miniature pig model by somatic cell nuclear transfer (SCNT) for studying atherosclerosis. The human apolipoprotein(a) (apo(a)) genes were transfected into kidney epithelial cells derived from a male and a female piglet. Male cells were used as donors initially, and 275 embryos were transferred to surrogates. Three offspring were delivered, and the production efficiency was 1.1% (3/275). Serial female cells were injected into 937 enucleated oocytes. Eight offspring were delivered (production efficiency: 0.9%) from surrogates. One male and 2 female transgenic miniature pigs matured well. Lipoprotein(a) was found in the male and one of the female transgenic animals. These results demonstrate successful production of human apo(a) transgenic NIBS miniature pigs by SCNT. Our goal is to establish a human apo(a) transgenic NIBS miniature pig colony for studying atherosclerosis.     

人DAF基因在转基因小鼠中的遗传与表达

为研究人ＤＡＦ基因在小鼠体内遗传与表达的规律,从质粒ｐＳＦＦＶ－ＤＡＦ分离出一段包含人ＤＡＦ基因的ＤＮＡ片段。采用受精卵显微注射法建立转人ＤＡＦ基因小鼠。提取出生小鼠的染色体ＤＮＡ,经Ｄｏｔ－ｂｌｏｔ与Ｓｏｕｔｈｅｒｎ－ｂｌｏｔ杂交相结合确定首建转基因小鼠,并经Ｄｏｔ－ｂｌｏｔ杂交研究人ＤＡＦ基因在转基因小鼠体内的遗传特征,Ｎｏｒｔｈｅｒｎ杂交确定其表达情况。小鼠受精卵经基因导入后,共生出２４只小鼠,其中４只被确定为首建转基因小鼠,整合率为１５％,在首建转基因小鼠两两交配生出的Ｆ１代小鼠中分别有７０％和７５％继续携带人ＤＡＦ基因。首建转基因小鼠中有１只小鼠在ＲＮＡ水平表达了人ＤＡＦ基因。可见,人ＤＡＦ基因整合入小鼠基因组中,并能够稳定遗传及表达。     

Mouse zygotes with one diploid pronucleus formed as a result of ICSI can develop normally beyond birth

A mouse spermatozoon was injected into mouse secondary oocytes (ICSI) in the vicinity of the metaphase spindle. In 22% of oocytes injected successfully, the maternal chromatin (the haploid chromatids formed after the second meiotic division) and paternal chromatin (from the sperm nucleus) were surrounded by a common nuclear envelope to form one diploid bi-parental pronucleus. However, the use of spermatozoa in which BrdU had been incorporated into DNA during spermatogenesis revealed, that maternal and paternal chromatin occupied two separate compartments within the one pronucleus. In the living state, the diploid pronucleus could be distinguished from a haploid one by its distinctly larger size and by a greater number of "nucleolus-like bodies"-criteria confirmed karylogically at the 1st cleavage division. Such zygotes with one diploid pronucleus were able to develop in vitro into blastocysts as often as those with two haploid pronuclei [11/29 (38%) vs. 14/35 (40%)]. Seventy nine 2-cell embryos developing in vitro from zygotes with one diploid pronucleus were transplanted to the oviducts of pseudopregnant recipients: two females had six foetuses when killed on the 17th day, and two females gave birth to nine young, eight of which survived and developed into normal fertile animals.     

Expression of the gene of interest fused to the EGFP-expressing gene in transgenic mice derived from selected transgenic embryos

The present paper describes the expression of a target fusion gene, WAP/hGH fused to the EGFP-expressing gene in transgenic mice derived from the transfer of transgenic embryos selected because of their expression of enhanced green fluorescent protein (EGFP). The 6.7-kb fusion gene was microinjected as a single cassette gene construct into the pronuclei of mouse zygotes. The surviving embryos were cultured and were classified according to the EGFP expression patterns at the morula or blastocyst stage. After the transfer of embryos with uniform-expression or mosaic-expression of EGFP, transgenesis occurred in 85.7% to 86% or 44.1% to 44% of the pups, respectively. No transgenic pups were derived from EGFP negative embryos. In the transgenic females, EGFP was ubiquitously expressed under the control of the CAG promoter, and hGH was expressed under the control of the WAP promoter in an appropriate fashion: hGH was secreted into the milk of lactating transgenic females. The presence or absence of the expression of EGFP coincided with that of the hGH gene in the transgenic mice. The present cassette gene construct is a useful example for circumventing the routine analyses of DNA and RNA required for the generation and maintenance of transgenic lines.     

A comparison of mouse and rabbit embryos for the production of transgenic animals by pronuclear microinjection

Procedures for the production of transgenic animals have low overall efficiency. To evaluate factors responsible for low efficiency, zygotes of two species, varying intensities of microscope light, different qualities of injection pipettes, and six different genes were tested for their influence on the efficiency of pronuclear gene injection for the production of transgenic rabbits and mice. Rabbit zygotes were less sensitive to mechanical manipulation during injection than mouse zygotes. Exposing zygotes to a microscope light intensity of 5550 lx significantly reduced their cleavage rate, while a lower intensity (2280 lx) did not. Using pipettes with a filament for pronuclear gene injection of mouse zygotes resulted in a higher cleavage rate of zygotes after injection than when pipettes were used without filament (30.3 vs 20.6%). Implantation rates varied between 2.9% (HB72CAT) and 23.1% (ts 58-2) depending on the gene used. No transgenic animals were obtained after injection of uteroglobin-CAT-hybrid genes (B2B3UGCAT, HB72CAT), while all other genes used (UG 11.8, UGTAg, RSV lacZ, ts 58-2) resulted in transgenic embryos, fetuses, and newborn animals.     

High-level expressing YAC vector for transgenic animal bioreactors

The position effect is one major problem in the production of transgenic animals as mammary gland bioreactors. In the present study, we introduced the human growth hormone (hGH) gene into 210-kb human alpha-lactalbumin position-independent YAC vectors using homologous recombination and produced transgenic rats via microinjection of YAC DNA into rat embryos. The efficiency of producing transgenic rats with the YAC vector DNA was the same as that using plasmid constructs. All analyzed transgenic rats had one copy of the transgene and produced milk containing a high level of hGH (0.25-8.9 mg/ml). In transgenic rats with the YAC vector in which the human alpha-lactalbumin gene was replaced with the hGH gene, tissue specificity of hGH mRNA was the same as that of the endogenous rat alpha-lactalbumin gene. Thus, the 210-kb human alpha-lactalbumin YAC is a useful vector for high-level expression of foreign genes in the milk of transgenic animals.