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.     

人促血小板生成素在转基因小鼠乳腺中定位表达的研究(英文)

通过转基因动物乳腺生物反应器大规模生产药用蛋白质已成为现代生物技术新的生长点之一。为研制表达人促血小板生成素的哺乳动物生物反应器的转基因小鼠模型,本论文以小鼠乳清酸蛋白 (mWAP) 基因5挾说骺厍团-s1-酪蛋白基因3挾说骺厍魑鹘谠菇擞糜诒泶锶舜傺“迳伤氐娜橄僮橹匾煨员泶镌靥錺WAPTPO(Fig.1)。通过常规显微注射的方法把mWAP启动子指导的hTPO表达载体导入小鼠受精卵,获得出生小鼠16只。经PCR检测,有6只为转基因阳性(Fig.2)。G0代小鼠中转基因整合率为37.5% (6/16),用ELISA方法在G0代转基因雌鼠的乳汁中检测了促血小板生成素的表达,表达量在0.8 mg/mL以上(Table 1)。这些结果表明我们已建立了乳腺表达hTPO 的转基因小鼠模型,为以后大型家畜乳腺生物反应器的研制提供了科学依据。     

Expression of microinjected reporter gene lacZ during first cleavages of rabbit embryos

The growing use of reporter genes in a model transgenic system has been a fundamental approach of biology, but the strategy of transgenic embryo selection prior to transfer to foster mothers may greately increase the efficiency of transgenic livestock production. This study was conducted to assess the possibility of beta-galactosidase (beta-gal)-labeled transgenic rabbit embryo production. Rabbit zygotes were obtained from superovulated females after mating. Zygotes were microinjected into male pronuclei with pCMV-lacZ or SV40-lacZ constructs; while some embryos were co-injected with the scaffold attachment sequences--SAR. Embryos from control non-injected and microinjected groups were cultured in vitro. After 24, 48, 72, or 96 h of culture the embryos were stained with X-gal for beta-galactosidase. Transgenic embryos produced by pronuclear injection showed a discrete pattern of beta-galactosidase expression. The percentage of transgenesis with pCMV-lacZalone was 1.5, but with SAR sequences it increased to 4.2. In the case of SV40-lacZ construct, the efficiency of transgenesis was 2.3% and 4.1%, respectively. The mosaicism was 66.7% for all embryos injected with both constructs with or without SAR. The highest numbers of 100%-transgenic (non-mosaic) embryos were found in the group co-injected with SV40-lacZ and SAR. Transgenesis was seen as early as 24 h after injection, in four-cell embryos. Most of the microinjected embryos showed delayed development as compared with control. It was concluded that lacZ may serve as a reliable reporter for early transgenic embryo selection in order to produce transgenic animals.     

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     

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.     

双原核注射提高转基因的整合效率(英文)

本文提出了受精卵双原核注射方法,利用共注射具有部分同源区的两个片段鼠乳消酸蛋白基因 (WAP) 调控序列控制下的人粒细胞集落刺激因子 (G-CSF) (Fig.1),通过PCR和Southern Blot检测了8细胞期外源基因的整合(Fig.2)。结果表明,与单一原核注射相比较,双原核注射转基因的整合率由单一原核注射的48.68%提高至62.65%(Table 1)。这为转基因动物建立,提高外源基因整合率提供了新途径。     

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.     

Direct comparison between ICSI-mediated DNA transfer and pronuclear DNA microinjection for producing transgenic rats.

Production efficiency of transgenic rats was compared directly between the routine pronuclear microinjection of exogenous DNA solution (PNMI-Tg method) and the ooplasmic injection of sperm cells exposed to exogenous DNA solution (ICSI-Tg method) using six DNA constructs. The overall production efficiency per treated oocyte in the ICSI-Tg method (mean 1.1%, range 0.2 to 3.1%) was similar to that in the PNMI-Tg method (mean 1.1%, range 0 to 2.4%). An advantage of the ICSI-Tg method in the production of transgenic rats is noted in cases in which a low yield of pronuclear zygotes is an inevitable fate of the rat strain.     

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.     

Transgenic mice generated by pronuclear injection of a yeast artificial chromosome.

Transgenic mice have become invaluable for analysing gene function and regulation in vivo. However, the size of constructs injected has been limited by the cloning capacity of conventional vectors, a constraint that could be overcome with yeast artificial chromosomes (YACs). We investigated the feasibility of making transgenic mice with YACs by pronuclear injection of a small YAC carrying a gene encoding tyrosinase. Use of a vector with a conditional centromere allowed fifteenfold amplification of the YAC in yeast and its recovery in high yield. The albino phenotype of the recipient mice was rescued demonstrating the correct expression of the tyrosine gene from the construct. Furthermore, the telomeric sequences added by the yeast integrated into the mouse genome and did not reduce efficiency of integration. Using this technique future experiments with longer YACs will allow the expression of gene complexes such as Hox and the globin gene clusters to be analysed in transgenic animals.     

Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein

The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9–FokI. We then applied the optimized conditions to zygotes, delivering Cas9 as either mRNA or protein. We found that Cas9 nucleo-protein complex promotes highly efficient, multiplexed targeting of circular constructs containing reporter genes and floxed exons. This approach allows for a one-step zygote injection procedure targeting multiple genes to generate conditional alleles via homologous recombination, and simultaneous knockout of corresponding genes in non-targeted alleles via non-homologous end joining.     

血红素加氧酶-1显性负性突变体转基因小鼠模型的建立与鉴定

目的建立血红素加氧酶-1(heme oxygenase-1,HO-1)显性负性突变体G143H转基因小鼠模型。方法通过SalI/DraI酶切pCAGGHO-1G143H转基因表达载体,纯化回收HO-1G143H表达盒片段;通过显微注射把表达目的基因的DNA片段导入FVB小鼠受精卵原核,并移植给同期发情的假孕受体母鼠,获得子代小鼠;用PCR对子代鼠尾DNA进行鉴定,并用Southern blot对结果做进一步验证;通过RT-PCR、免疫组化和Western blot方法检测HO-1基因的表达。结果表达盒回收片段正确;假孕鼠出生的17只子代小鼠共有3只阳性,均为雄性;RT-PCR、免疫组化和Western blot结果表明,阳性小鼠体内的HO-1 mRNA与蛋白表达水平增高。结论成功建立HO-1显性负性突变体G143H表达的转基因小鼠,该模型为研究HO-1在体内的作用机制奠定了基础。     

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.     

Subcellular distribution of mitochondrial ribosomal RNA in the mouse oocyte and zygote

Mitochondrial ribosomal RNAs (mtrRNAs) have been reported to translocate extra-mitochondrially and localize to the germ cell determinant of oocytes and zygotes in some metazoa except mammals. To address whether the mtrRNAs also localize in the mammals, expression and distribution of mitochondrion-encoded RNAs in the mouse oocytes and zygotes was examined by whole-mount in situ hybridization (ISH). Both 12S and 16S rRNAs were predominantly distributed in the animal hemisphere of the mature oocyte. This distribution pattern was rearranged toward the second polar body in zygotes after fertilization. The amount of mtrRNAs decreased around first cleavage, remained low during second cleavage and increased after third cleavage. Staining intensity of the 12S rRNA was weaker than that of the 16S rRNA throughout the examined stages. Similar distribution dynamics of the 16S rRNA was observed in strontium-activated haploid parthenotes, suggesting the distribution rearrangement does not require a component from sperm. The distribution of 16S rRNAs did not coincide with that of mitochondrion-specific heat shock protein 70, suggesting that the mtrRNA is translocated from mitochondria. The ISH-scanning electron microscopy confirms the extra-mitochondrial mtrRNA in the mouse oocyte. Chloramphenicol (CP) treatment of late pronuclear stage zygotes perturbed first cleavage as judged by the greater than normal disparity in size of blastomeres of 2-cell conceptuses. Two-third of the CP-treated zygotes arrested at either 2-cell or 3-cell stage even after the CP was washed out. These findings indicate that the extra-mitochondrial mtrRNAs are localized in the mouse oocyte and implicated in correct cytoplasmic segregation into blastomeres through cleavages of the zygote.     

First cell cycle of zygotes of the mouse derived from oocytes aged postovulation in vivo and fertilized in vivo

This paper describes an analysis of the first cell cycle of mouse oocytes aged postovulation and fertilized in vivo. For this purpose, we developed a procedure for inducing ovulation in vivo that allows accurate timing of ovulation. The method is based on a luteinizing hormone (LH)-releasing hormone (LHRH) administration at proestrus. This ovulation procedure had no detectable effect on the rate of ovulation or postimplantation embryonic death. We used this method of ovulation induction in an analysis of the separate stages of the first cell cycle of in vivo fertilized postovulation aged oocytes. All stages assessed were shorter in aged oocytes (12 hr postovulation) than in zygotes from unaged oocytes (1 hr postovulation): 1) the time interval between insemination and penetration of the aged oocytes was 1.5 hr shorter than the time interval of the unaged oocytes; 2) pronuclear formation in the fertilized aged oocytes was somewhat quicker than pronuclear formation in fertilized unaged oocytes; 3) in zygotes from aged oocytes, the time between formation of pronuclei and the pronuclear membrane breakdown was 1 hr shorter than in zygotes from unaged oocytes; 4) the first cleavage division was 3 hr advanced in zygotes from aged oocytes compared with the moment of the first cleavage division in zygotes from unaged oocytes. We also determined the glutathione (GSH) content of unaged and aged oocytes to investigate a possible relationship between the rate of pronuclear formation and GSH. The level of GSH was two times lower in oocytes aged postovulation for 12 hr than in unaged oocytes.2+ level of GSH in fertilized, unaged oocytes was half that in     

Generation of an Fsp1 (fibroblast‐specific protein 1)‐Flpo transgenic mouse strain

Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases (Flp, Flpe, and Flpo) bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1‐Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of Fsp1 (fibroblast‐specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1‐Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype.     

Spermatogenic Cell-Specific Gene Mutation in Mice via CRISPR-Cas9

Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals.However,conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process,restricting rapid study of the gene function in vivo.CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique,which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes.Here,we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step.We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells.We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene.Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally.Consistently,male progeny from female founders were infertile and females could transmit the transgenes to the next generation.Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern.     

Kinetics of pronuclear development and the effects of vector type and timing of injection on the efficiency of gene transfer into rhesus macaque embryos

A series of experiments was performed to determine the dynamics of pronuclear development as well as the efficiency of either adenovirus-associated (AAV) or lentivirus-derived vectors to introduce a green fluorescent protein (GFP) reporter gene into rhesus macaque (Macaca mulatta) embryos. Assessment of pronuclear development at various times after fertilization revealed that the appearance of pronuclei was determined by the presence of the first and the timing of the second polar body. The dynamics of pronuclear formation was a significant determinant of whether an oocyte reached the blastocyst stage, however, when the percentage of blastocysts were based on the number of zygotes, the timing of the appearance of polar bodies did not appear to have any effect on subsequent development. Injection of different AAV-derived vectors showed that the serotype of the vector did not affect development or the proportion of transgenic embryos. Moreover, all putative transgenic embryos proved to be expression mosaics. Injection of embryos with lentiviral vectors showed that timing of injection (before or after fertilization) had no effect on subsequent transgene expression, but that the type of reporter gene determined post-injection development and rate of transgenesis. The transfer of embryos following injection of a lentiviral vector into three recipients resulted in one pregnancy which was lost during the second trimester. Analysis of fetal tissues showed ubiquitous presence of the transgene and GFP expression in all tissues examined. These results show that lentivirus-derived vectors can efficiently transform rhesus embryos and are suitable for the generation of transgenic rhesus monkeys.     

Developmental patterns of zygotes from transgenic female mice with elevated tissue glutathione

Zygotes were collected from transgenic mice overexpressing glutathione synthetase to test the hypothesis that such zygotes are more capable of developing in suboptimal culture media than zygotes from non-transgenic (control) mice. The effects of injection of donor mice with gamma-glutamylcysteinyl ethyl ester (gamma-GCE) on embryonic development were also investigated. In addition, the effects of genetic background (i.e., transgenic vs. non-transgenic) and injection with gamma-GCE on developmental capacity in kSOM were studied after exposure of zygotes to diamide (0.01 mM, for 30 min). When cultured in modified Medium 16 significantly more pronuclear ova from transgenic females reached the morula (M) and early blastocyst (EB) stages than embryos derived from control mice. Genetic background significantly affected the proportions of embryos reaching 4- to 16-cell, M and EB stages during culture in modified Whitten's medium (WM); more zygotes collected from transgenic than from control mice developed. The injection of experimental mice with gamma-GCE significantly increased proportions of zygotes developing to M and EB stages in WM. Following exposure to diamide and subsequent culture in kSOM significantly more zygotes collected from transgenic mice reached the 4- to 16-cell stages than those from control females; a significant positive effect on developmental capacity was also seen after injection of donor mice with gamma-GCE. When cultured in suboptimal conditions, zygotes derived from transgenic mice overexpressing glutathione synthetase were more capable of developing than zygotes of non-transgenic control females. Zygotes from the transgenic mice also exhibited greater capacity to withstand toxic exposure to diamide. Present data suggest that commonly known strain differences in preimplantational development in vitro may reflect differences in the synthesis and/or metabolism of glutathione. J. Exp. Zool. 286:173-180, 2000.     

The effects of spermatozoal irradiation with X-rays on chromosome abnormalities and on development of mouse zygotes after delayed fertilization

The chromosome complements of zygotes derived from oocytes aged post ovulation and fertilized in vivo with X-ray-irradiated sperm were studied. Ovulation was induced by an injection of luteinizing hormone-releasing hormone (LHRH) at pro-estrus and fertilization was achieved by artificial insemination at 13 h and 24 h after LHRH in order to obtain embryos from unaged and aged (12 h post-ovulation) oocytes respectively. Post-ovulatory aging prior to fertilization did not significantly affect the percentage of zygotes with irradiation-induced chromosome abnormalities. However, post-ovulatory aging had a negative effect on the morphology of male as well as female pronuclear chromosomes of the first cleavage metaphase. When fertilized with control spermatozoa this effect was apparent in both the male and the female pronucleus. When unaged oocytes were fertilized with X-irradiated spermatozoa chromosome morphology was also adversely affected in both pronuclei. In zygotes from aged oocytes, there was an extra negative effect of X-rays on the male pronuclear chromosomes only. After fertilization with X-irradiated sperm 27% of zygotes from aged oocytes were arrested at interphase compared to 7% from unaged oocytes. We suggest that post-ovulatory aging and X-rays affect the male and female pronuclear chromatin structure after fertilization. These chromatin alterations could interact with DNA lesions induced in the spermatozoa prior to fertilization, such that development to first cleavage can be blocked.