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     

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.     

嗜铬颗粒蛋白A基因的反义转基因小鼠模型的建立及该基因启动子的组织特异性

构建小鼠嗜铬颗粒蛋白A（Chromogranin,A,CGA)基因的反义DNA载体pGAS1C－lacZ。用电穿孔的方法将该载体转化大鼠肾上腺髓质细胞瘤细胞系PC－12,X－Gal染色后证明位于CGA基因启动子下游的报告基因lacX已经表达。用限制性内切酶除去载体的质粒骨架后,显微注射入供体昆明小鼠的受精卵中,随后将注射过DNA的受精卵移植入假母的输卵管中,完成正常的胚胎发育。用PCR的方法筛选假母产下的小鼠,得到CGA基因反义RNA基因首建鼠14只。将首建鼠分别与正常昆明鼠交配,产生后代。取首建鼠的肾上腺进行X－Gal染色,组织用于石蜡切片,根据各鼠肾上腺切片的蓝色深浅判定转入基因量的高低,筛选到两只表达量高的首建鼠,留下它们的后代。取转基因鼠的各种组织用于X－Gal染色,发现报告基因在肾上腺、胰腺的胰岛中有表达,而在肌肉、脂肪组织中无表达,说明CGA基因的启动子具有神经内分泌组织特异性。     

Transgenic mice expressing a chimaeric anti-E. coli immunoglobulin α heavy chain gene

To induce constitutive immunity against a pathogenic strain ofEscherichia coli (K99), a rearranged immunoglobulin (Ig) heavy chain (HC) gene was constructed. Because the route ofE. coli infection is enteric, an IgA transgene was desirable. A chimaeric gene construct was cloned that coded for a HC that recognized a specificE. coli pilus antigen. The construct comprised a gene promoter, murine VDJ, and bovine -HC constant region. Following microinjection of the HC construct into murine zygotes, of 50 liveborn mice, three were identified as transgenic. In all three transgenic founders, transgene-encoded mRNA expression was detected by northern blot. The transgenic founders were analysed for transgene-encoded RNA expression in splenic tissue before and after challenge with pathogenicE. coli. Founder 4-3 expressed transgene-encoded RNA both before and after challenge; expression was detected in the other two founders only post-challenge. As no differences were found when sera were analysed for bovine IgA in control and transgenic mice, protein expression was assessed by challenge of HC founders with K99E. coli by gavage. Control mice challenged with K99E. coli were moribund within 24 h post-gavage, but there was no observable affect in the three transgenic founders. Unfortunately, after obtaining offspring from all founders, no transgenic offspring were identified (0/108). The low yield of transgenic founders, coupled with the apparent germ-line mosaicism may point to either mechanical or critical developmental anomalies. However, the production of transgenic mice harbouring an Ig HC gene construct confirmed that an Ig transgene coding for an antibody to an animal pathogen could function in a tissue-specific and protective manner in a mammalian system.     

A 3,387?bp 5′-flanking sequence of the goat alpha-S1-casein gene provides correct tissue-specific expression of human granulocyte colony-stimulating factor (hG-CSF) in the mammary gland of transgenic mice

A new expression vector containing the 1,944 bp 5'-flanking regulatory region together with exon 1 and intron 1 of the goat alpha-S1-casein gene (CSN1S1), the full-sized human granulocyte colony-stimulating factor gene (hGCSF) and the 3'-flanking sequence of the bovine CSN1S1, was created. The vector DNA was used for generation of four mouse transgenic lines. The transgene was integrated into chromosomes 8 and 12 of two founders as 2 and 5 copies, respectively. Tissue-specific secretion of hG-CSF into the milk of transgenic mice was in the range of 19-40 μg/ml. RT-PCR analysis of various tissues of the transgenic mice demonstrated that expression of hGCSF was detected in only the mammary gland in the progeny of all founders. Moreover, cells were shown to be positive for hG-CSF by immunofluorescent analysis in the mammary glands but not in any other tissues. There were no signs of mosaic expression in the mammary gland. Trace amounts of hG-CSF were detected in the serum of females of two transgenic lines during lactation only. However, no transgenic mice showed any changes in hematopoiesis based on the number of granulocytes in blood. Immunoblotting of hG-CSF in the milk of transgenic mice revealed two forms, presumably the glycosylated and non-glycosylated forms. The hematopoietic activity of hG-CSF in the milk of transgenic females is comparable to that of recombinant G-CSF. In general, the data obtained in this study show that the new expression vector is able to provide correct tissue-specific expression of hG-CSF with high biological activity in transgenic mice.     

Positive regulatory elements (HF-1a and HF-1b) and a novel negative regulatory element (HF-3) mediate ventricular muscle-specific expression of myosin light-chain 2-luciferase fusion genes in transgenic mice.

The cardiac myosin light-chain 2v (MLC-2v) gene has served as a model system to identify the pathways which restrict the expression of cardiac muscle genes to particular chambers of the heart during cardiogenesis. To identify the critical cis regulatory elements which mediate ventricular chamber-specific expression of the MLC-2v gene in the in vivo context, a series of transgenic mice which harbor mutations in putative MLC-2 cis regulatory elements in a 250-bp MLC-2-luciferase fusion gene which is expressed in a ventricular chamber-specific fashion in transgenic mice were generated. These studies demonstrate that both components of HF-1 (HF-1a and HF-1b/MEF-2) are required to maintain ventricular chamber-specific expression and function as positive regulatory elements. Mutations in another conserved element (HF-2) are without statistically significant effect on ventricular chamber expression. Transgenics harboring mutations in the E-box site also displayed significant upregulation of reporter activity in the soleus, gastrocnemius, and uterus, with a borderline effect on expression in liver. Mutations in another conserved element (HF-3) result in a marked (> 75-fold) upregulation of the luciferase reporter activity in the soleus muscle of multiple independent or transgenic founders. Since the HF-3 mutations appeared to have only a marginal effect on luciferase reporter activity in liver tissue, HF-3 appears to function as a novel negative regulatory element to primarily suppress expression in muscle tissues. Thus, a combination of positive (HF-1a/HF-1b) and negative (E-box and HF-3) regulatory elements appear to be required to maintain ventricular chamber-specific expression in the in vivo context.     

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.     

Rapid generation of dendritic cell specific transgenic mice by lentiviral vectors

Dendritic cell (DC) specific transgenic mice are a most important model for investigating dendritic cell functions in vivo. Recently, lentivirus mediated gene transfer has become a powerful and convenient method for generation of transgenic mice. We cloned a 1.2 kb CD11c promoter and constructed a lentiviral vector, which efficiently drove DC-specific expression in vitro. After microinjection of purified virus into the perivitelline space of single-cell embryo, more than 80% newborn mice were transgenic and 7 F0 founders were rapidly generated in 2 months. GFP was strictly expressed in CD11c+ cells in spleens, thymus and lymph nodes of the transgenic mice. Importantly, the physiological characteristics and functions of DCs in the transgenic mice were not altered by the specific expression. These results indicate that this vector could be used to rapidly prepare DC-specific transgenic mice. Thus, this lentiviral vector system may provide a convenient and useful tool to study the properties of DCs in vivo.     

Expression of CD80 promoter in transgenic mice

CD80 is a very potent co-stimulatory factor which is required for complete T-cell activation. Here, we use transgenic mice as a tool to map the promoter of the CD80 gene. We engineered three different CD80 promoter driven luciferase transgenes: -3084, -1073 and -215. With these transgenes, we have generated three groups of transgenic mice. Our results showed that the -3084 CD80 promoter/luciferase transgene was sufficient to confer tissue-specific expression of the CD80 gene. When the promoter sequence was deleted to -1073, the normal tissue-specific expression was lost. A brain-specific element was mapped between -1073 nt and -215 nt. This element caused up to ninefold higher expression of the CD80 promoter/luciferase in brain tissue of -1073 CD80 promoter/luciferase transgenic animals as compared to -3084 CD80 promoter/luciferase transgenic animals. In contrast to results with a cell culture system, little luciferase activity was detected in -215 CD80 promoter/luciferase transgenic animals.     

Dicer1转基因小鼠模型的建立

目的建立Dicer1转基因小鼠模型。方法构建pcDNA3.1-Dicer1转基因构件,经酶切、纯化后通过显微注射方法导入BDF1小鼠受精卵原核并移植到同期受孕的ICR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,通过免疫组化检测Dicer1基因表达。结果显微注射172枚卵,移植119枚卵于3只受体输卵管中,2只怀孕,共产仔15只,经PCR检测获得6只阳性鼠,Southern检测6只均为阳性。对Southern检测阳性转基因小鼠子代进行RT-PCR检测和免疫组化分析证明Dicer1基因在肝脏、肾脏、肺内均有表达。对腹腔肿胀的转基因阳性1号鼠解剖发现肝脏、脾脏明显增大,胚胎发育异常。结论成功建立Dicer1基因表达的转基因小鼠模型,该模型为进一步研究DICER1基因功能及miRNA的表达及功能等奠定基础。     

增强子驱动的Cre转基因小鼠的构建及Cre基因的表达鉴定

目的:探索将增强子应用于构建Cre转基因小鼠品系,为以条件基因敲除为基础的基因功能研究提供更多的工具。方法:通过PCR方法从小鼠的细菌人工染色体扩增UH增强子片段,构建含有Hsp68基础启动子、增强子UH、Cre重组酶基因和SV40 polyA的转基因载体pLW400,将3.3 kb的转基因片段通过显微注射导入小鼠受精卵;为了检测Cre在转基因小鼠中的表达,将转基因一代小鼠与纯合子ROSA26报告小鼠(R/R)交配,收集第14 d胚胎期(E14)的舌组织进行LacZ染色检测鉴定。结果:经鉴定,31只子代小鼠中有6只携带外源基因,整合率为19.4%;与R/+对照相比,E14期的双基因型Cre,R/+舌组织为阳性结果(蓝色)。这表明Cre基因在转基因小鼠舌组织内得到表达,并在体内介导ROSA26基因座loxP位点间的重组,且有效删除了2个loxP之间的片段,从而启动了LacZ基因的表达。结论:构建了UH增强子-Hsp68Cre的转基因小鼠,在舌肌中特异表达Cre基因,提示增强子可以被选择应用于Cre转基因小鼠的构建;为舌肌的发育和再生研究奠定了基础。     

多巴胺D5受体转基因小鼠的建立

目的建立人多巴胺D5受体突变基因F173 L（D5F173L）,S390G（D5S390G）及正常人D5基因（hD5 WT）的转基因小鼠,利用该转基因动物模型来研究D5受体在原发性高血压中的发病机制。方法利用显微注射的技术将插入CMV启动子下游的D5F173L,D5S390G及hD5 WT基因的转基因载体注射入C57BL/6小鼠体内,建立多巴胺D5F173L,D5S390G及hD5 WT的转基因小鼠。通过PCR鉴定转基因小鼠的基因型。利用Western Blotting方法鉴定该受体蛋白在肾脏的表达情况。使用智能无创血压测量仪测量转基因小鼠的血压值。结果分别建立了多巴胺D5F173L,D5S390G及hD5 WT转基因C57BL/6小鼠,Western Blotting方法鉴定结果显示,与非转基因C57BL/6小鼠比较,D5转基因小鼠D5受体在肾脏有较高的表达。3-6月龄D5 F173L转基因小鼠的收缩压、舒张压和平均动脉血压均明显高于多巴胺D5S390G及hD5 WT转基因小鼠（n=6-8,P〈0.05）。结论多巴胺D5受体在原发性高血压发病中具有重要作用,但作用机理还有待于进一步研究。     

心脏特异表达NOL3转基因小鼠的建立

目的建立心脏特异表达NOL3转基因小鼠,用于研究该基因在心肌病发病中的作用。方法Western blot检测小鼠NOL3表达谱。构建aMHC-NOL3表达载体,显微注射法建立NOL3转基因小鼠。PCR鉴定转基因鼠的基因型,心脏超声检测转基因及野生型小鼠心脏功能及几何构型。结果NOL3在1月龄野生型鼠心脏、脑、骨骼肌中的高表达,在心脏中的表达不随年龄而改变。通过转基因小鼠的筛选,得到了3个NOL3转基因品系,其中1个品系心脏NOL3蛋白表达量与野生型鼠相比明显增加。单转NOL3基因的小鼠心脏功能及几何构型与野生型小鼠相比无显著变化。结论成功建立了心脏特异表达NOL3转基因小鼠,为进一步和心肌病小鼠模型杂交,研究该基因在心肌病发病中的作用提供了工具。     

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,     

Rat synapsin 1 promoter mediated transgene expression in testicular cell types

Previous reports described the rat synapsin 1 promoter as primarily neuron selective. However, ectopic expression of a transgene under the rat synapsin 1 promoter was also detected in testis from some transgenic mouse lines. Here we investigate which cells within the testis express a transgene consisting of the rat synapsin 1 promoter fused with luciferase. Synapsin 1-luciferase expression vectors were introduced into HeLa cells, into TM3 cells derived from mouse testicular Leydig cells, and into one-cell embryos to make transgenic mice. Indirect immunofluorescence suggests that nontransfected TM3 cells do not express endogenous synapsin 1. TM3 stable transfectants, however, expressed luciferase under the direction of the synapsin 1 promoter, in both promoter orientations. HeLa cells displayed only low levels of activity. Transgenic mice carrying the synapsin 1-luciferase construct displayed high levels of luciferase activity in the brain, spinal cord, and testis. Enriched populations of prepuberal types A and B spermatogonia and adult Leydig cells, pachytene spermatocytes, and round spermatids prepared from transgenic mice all displayed substantial luciferase activity. Thus, the rat synapsin 1 promoter can mediate reporter gene expression in neurons and testicular cell types.     

Determination of the expression of fish antifreeze protein (AFP) in 7th generation transgenic mice tissues and serum

In this study, the presence of antifreeze protein (AFP) gene expression through successive generations in transgenic mice carrying the chimeric gene construct of the coding sequence for the AFP protein from ocean pout was investigated. AFP transgenic hemizygote mice were used for AFP gene expression. AFP genome expressions in transgenic mice were analyzed by Western blotting, and tissue location of AFP protein was shown by immunohistochemical and immunofluorescence techniques. Seventh transgenic mice from the established founders demonstrated the expression of AFP in organs such as the skin, oviduct, lung, kidney and liver tissues and serum except for the heart. Our results demonstrate successful expression of AFP gene products in several tissues and serum of transgenic mice, the association of in vivo expressed AFP protein, for the first time. These results indicate that the coding sequence for the AFP protein gene (ocean pout type III AFP gene) could be integrated and stably transcribed and expressed in the 7th generation of transgenic mice. In conclusion transgenic mouse lines would be a good model for the cryostudy of AFP and for the determination of AFP roles in several organs and tissues.