SHBG基因敲除小鼠模型的建立及其表型分析

目的:建立性激素结合球蛋白(SHBG)基因条件敲除小鼠模型,为探讨胎盘组织中SHBG在体内的生理功能及其与妊娠期糖尿病发病关系提供实验手段。方法:首先运用生物信息学手段确定小鼠SHBG基因组序列,构建SHBG打靶载体,以电穿孔方法将其导入小鼠ES细胞,筛选培养阳性ES细胞并行PCR鉴定,并将正确同源重组的ES细胞注射进小鼠囊胚,移入受体小鼠子宫;将获得的嵌合体小鼠与C57BL/6J小鼠交配,筛选后获得Flox小鼠,该小鼠与EIIa-Cre转基因小鼠杂交,子代多次自交获得SHBG全身基因敲除(SHBG~(-/-))的小鼠。结果:运用同源重组及ES细胞技术建立了SHBG基因的Flox小鼠,并利用Cre/Loxp重组酶系统建立了SHBG基因全身敲除小鼠模型,PCR方法从基因水平证明了SHBG基因Flox小鼠及SHBG基因全身敲除小鼠模型建立成功。对基因敲除鼠进行初步表型分析发现:SHBG基因全身敲除小鼠的生长发育与野生型小鼠相比无明显肉眼所见异常,SHBG基因全身敲除雌雄小鼠均具有生殖能力。结论:成功建立SHBG基因全身敲除小鼠模型,通过对基因敲除鼠进行初步表型分析,发现SHBG基因全身敲除小鼠外观上发育正常,为进一步研究SHBG在妊娠期糖尿病中的作用奠定了基础。     

小鼠新基因Ayu17-449的克隆及表达分析

在利用PU8捕获载体从小鼠ES细胞中寻找有关对发育起重要作用基因时,一阳性ES克隆编号为Ayu17-449被捕获,经过Southern blotting法证实捕获载体单一整合在Ayu17-449号ES细胞的基因组中。通过用5＇RACE法得到所捕获基因的一小段cDNA,在EST数据库中比对,得到一5523bp cDNA序列,在Celera数据库中它包含于两个相邻基因,根据这两个基因的mRNA设立了一系列的引物进行RT-PCR和测序,用这两个基因的不同片段分别作探针进行Northern blotting分析,确定这是一个RNA约9kb并编码1920个氨基酸的新基因（定名为Ayu17-449基因,其cDNA序列和编码蛋白序列发表在NCBI数据库,编号为DQ079067）。Northern blotting揭示Ayu17-449基因高度表达在小鼠的脑、肾脏、心脏、肺、肌肉和胃等组织。PU8捕获载体具有X-gal报告基因,能从蛋白表达水平揭示它所捕获的基因的表达模式。X-gal染色结果显示,Ayu17-449蛋白高度表达在小鼠的脑、肾脏、心脏等组织,与Northern blotting法的结果高度一致。X-gal染色切片结果进一步证明Ayu17-449蛋白主要表达在脑的神经细胞和肾脏近曲小管细胞中。Ayu17-449基因的编码蛋白在数据库（Scansite,http：//scansite．mit．edu/）做功能基团分析后,揭示其编码蛋白的N末端含有Granin基团,大量文献证实Granin基团具有参与激素的分泌的功能,显示Ayu17-449基因可能与激素的分泌有关。     

羊FSHR基因5′端转录启动调控区生物学特性

文章对小尾寒羊、滩羊和澳洲绵羊等繁殖性状不同的3种绵羊与排卵有关的FSHR基因5′端转录启动调控区进行了克隆和分析,通过对FSHR基因的15个转录调控元件序列进行比较,结果表明,羊不同品种FSHR基因的转录调控元件序列之间没有差异。这说明绵羊的品种与FSHR基因5′端转录启动调控区的相关性不强,排除了因转录调控元件突变而影响转录调节能力的可能性。      

RNAi技术在转基因动物中的应用

RNAi可以作为一种有效的工具用来产生转录后沉默的效果,从而抑制特定基因的表达,已经在线虫、果蝇、小鼠、大鼠等模式生物中得到成功应用。RNAi转基因小鼠的出现,使得在哺乳动物整体水平研究靶基因的敲低成为可能。文章以RNAi转基因小鼠为代表,就转基因载体的设计策略、基因敲除与基因敲低的比较、RNAi转基因动物的优势以及目前存在的缺陷等作一总结,并展望了RNAi转基因动物对功能基因组研究的贡献以及应用前景。

     

利用基因诱捕技术进行小鼠基因剔除的初步研究

对利用基因诱捕技术进行小鼠基因剔除做了初步的探索,为进一步应用该技术进行小鼠基因功能研究奠定了基础.利用基因诱捕载体转染小鼠ES细胞,获得了36株neo基因单拷贝整合的诱捕ES细胞,其中14株细胞表达有活性的β半乳糖苷酶.将3株诱捕ES细胞分别经显微注射引入到受体囊胚中,再植入假孕母鼠的子宫中使其发育成小鼠.两株细胞得到了程度不同的嵌合体小鼠,其中一株诱捕ES细胞整合至生殖系.利用质粒拯救实验获得了诱捕载体整合位点附近的基因组序列,通过序列比对发现被诱捕的基因可能是一个新基因.X-gal染色结果显示,该基因的表达局限于小鼠腹部及肢芽的部位.     

小麦条锈菌国际鉴别寄主Vilmorin23的抗条锈病基因YrV23微卫星标记

Vilmorin23是小麦条锈菌国际鉴别寄主和国际上重要抗源材料。采用SSR技术,利用由Vilmorin23为基因供体转育而成的小麦抗条锈近等基因系Taichung29*6/YrV23,选用YrV23所在2B染色体上的55对SSR引物,对Taichung29*6/ YrV23及其轮回亲本Taichung29和抗性基因供体Vilmorin23的基因组DNA进行PCR扩增和聚丙烯酰胺凝胶电泳分析。结果显示,引物Xwmc356在近等基因系与轮回亲本间扩增出特异性DNA片段,经F₂代群体150个抗、感单株检测证实,该片段位点与抗条锈病基因YrV23有连锁关系,遗传距离为9.4 cM。Xwmc356可作为抗条锈基因YrV23的SSR标记。      

联会复合体——原发无精症发病中的重要角色

联会复合体(synaptonemal complex,SC)是一种减数分裂特异性超分子蛋白质结构,与减数分裂I(改罗文)中同源染色体的凝缩、配对、重组和分离密切相关。近年来,联会复合体的研究取得了一系列重要的进展,包括在其组成成分和功能上的一些新发现。在小鼠不育模型中联会复合体及其编码基因的异常可引起精子发生障碍。更重要的是,联会复合体编码基因之一SCP3单个碱基缺失导致的无精症已在人类原发不育患者中得到证实。对联会复合体基因SCP1的进一步研究也正在进行之中。      

利用SRY基因和微卫星标记鉴定反刍动物性别

以反刍动物为研究对象,应用多重PCR技术扩增绵羊基因组中X、Y染色体上的4个微卫星标记和SRY基因, 根据基因型进行性别鉴定,试图通过一次DNA扩增同时提供性别鉴定和基因分型的信息。结果表明所设计SRY基因的引物具有高度特异性,是性别鉴定的主要依据,而Y染色体上的MCM158、MAF45两标记由于特异性不好,因此不适用于性别鉴定,对于X染色体上所选的两标记MILVET09和AE25只能进一步验证所鉴定的雄性个体。得出结论在被检个体中,能同时扩增出SRY基因、MCM158、MAF45,X染色体上MILVET09和AE25,且X染色体上的MILVET09、AE25基因型为纯合子的个体为正常的雄性;被检个体中只有Y染色体上MCM158、MAF45和X染色体上MILVET09、AE25的扩增产物,而没有SRY基因的扩增产物,则被检个体为雌性,且MILVET09、AE25的基因型对雌性个体的性别判断无影响。MCM158、MAF45两标记基因型不影响个体的性别鉴定结果。
     

陆地棉SUPERMAN类似锌指蛋白基因的克隆与表达分析

锌指蛋白是生物体内数量最多的转录调控因子,它在动植物的生长发育中都起到十分重要的作用。SUPERMAN类锌指蛋白只含有1个锌指结构。我们根据这类蛋白的保守结构域设计简并引物,通过RT-PCR从棉花中获得了3个这个家族成员的EST,得到1个锌指蛋白基因的全长序列,该基因的编码区长744 bp,编码长248个氨基酸的多肽,其氨基酸序列与GenBank中登录的一个拟南芥RBE蛋白有40%的同源性。此基因被命名为GZFP。它含有保守的锌指结构并在多肽链的C-端具有富含亮氨酸的保守结构域,GZFP含有核定位信号并且没有内含子。GZFP基因在棉花花蕾、子房、花瓣和根中的表达量要高于木质部、韧皮部、叶片、纤维和种子。GZFP基因的表达量很低,在GenBank中没有任何和它同源的EST序列存在。对GZFP 5′侧翼区进行分析发现有数个花粉和根特异表达相关元件,4个与Dof蛋白作用的核心序列,4个与光诱导相关的元件。      

线粒体钾通道Kcna3基因敲除小鼠初步制备

目的 为观察线粒体钾通道在缺血再灌注(I/R)心肌损伤中的作用,探讨其和心衰的关系,制备基因敲除小鼠模型以探讨钾通道单分子作用.方法 用BAC载体制备同源重组载体,对129小鼠胚胎干细胞(ES)打靶筛选后,显微注射至C57 BL/6J小鼠囊胚获得嵌合小鼠.经尾基因组DNA PCR鉴定和测序,鉴别杂合子小鼠.结果 在40只灰色小鼠中初步鉴定出Kcna3+/-基因型F1小鼠8只.结论 在国内首先用ES同源重组基因打靶方法,成功育成Kcna3基因敲除鼠杂合子,为下一步获得纯合子鼠奠定了基础.对进一步用钾离子通道病模型研究心肌保护病理生理机制和药物筛选具重要意义.     

Cloning and Expression Analysis of a Murine Novel Gene, Ayu17-449

We used gene trapping vector PU8 to search some interesting genes which play important roles in mouse development from murine ES cells. One positive ES colony termed Ayu17-449 was trapped. Its partial cDNA was obtained by using 5′ RACE method. It is homologous to a 5523 bp cDNA fragment (GI: 20879412) in EST database. Further analysis of the 5523 bp cDNA sequence in Celera mouse gene database showed that it overlaps two genes. We designed serials of DNA primers according to the mRNAs of these two genes for RT-PCR and Northern blotting analysis, and identified a novel RNA about 9 kb (we named it as Ayu17-449) encoding 1920 aa. This gene is expressed highly in the brain, kidney, heart, lung, muscle and stomach. The expressed protein contains a Granin motif on its N-terminus, showing that this gene may be involved in hormone secretion.     

Characterization of Ayu17-449 gene expression and resultant kidney pathology in a knockout mouse model

The gene trap technique is a powerful approach for characterizing and mutating genes in the mouse. We used this method to identify a mouse gene of unknown function and to establish a mutant mouse line. We subsequently identified one gene, denoted Ayu17-449, on mouse chromosome 3 that comprised 14 exons encoding 1920 amino acids with a granin motif in its N-terminal sequence. In adult mice, this gene was highly expressed in the brain, heart, lung, muscle, stomach, and kidney. The insertion of a trap vector into the second intron of this gene resulted in the null mutation. Homozygous mice for these mutation died by 1 day after birth. Mutant mice showed a loss of acidic granules in the proximal convoluted tubules of the kidney. Our data demonstrates that Ayu17-449 is important for mouse survival.     

EUCOMM--the European conditional mouse mutagenesis program.

Functional analysis of the mammalian genome is an enormous challenge for biomedical scientists. To facilitate this endeavour, the European Conditional Mouse Mutagenesis Program (EUCOMM) aims at generating up to 12 000 mutations by gene trapping and up to 8000 mutations by gene targeting in mouse embryonic stem (ES) cells. These mutations can be rendered into conditional alleles, allowing Cre recombinase-mediated disruption of gene function in a time- and tissue-specific manner. Furthermore, the EUCOMM program will generate up to 320 mouse lines from the EUCOMM resource and up to 20 new Cre driver mouse lines. The EUCOMM resource of vectors, mutant ES cell lines and mutant mice will be openly available to the scientific community. EUCOMM will be one of the cornerstones of an international effort to create a global mouse mutant resource.     

Database for exchangeable gene trap clones: Pathway and gene ontology analysis of exchangeable gene trap clone mouse lines

Gene trapping in embryonic stem (ES) cells is a proven method for large‐scale random insertional mutagenesis in the mouse genome. We have established an exchangeable gene trap system, in which a reporter gene can be exchanged for any other DNA of interest through Cre/mutant lox‐mediated recombination. We isolated trap clones, analyzed trapped genes, and constructed the database for Exchangeable Gene Trap Clones (EGTC) [ http://egtc.jp ]. The number of registered ES cell lines was 1162 on 31 August 2013. We also established 454 mouse lines from trap ES clones and deposited them in the mouse embryo bank at the Center for Animal Resources and Development, Kumamoto University, Japan. The EGTC database is the most extensive academic resource for gene‐trap mouse lines. Because we used a promoter‐trap strategy, all trapped genes were expressed in ES cells. To understand the general characteristics of the trapped genes in the EGTC library, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathway analysis and found that the EGTC ES clones covered a broad range of pathways. We also used Gene Ontology (GO) classification data provided by Mouse Genome Informatics (MGI) to compare the functional distribution of genes in each GO term between trapped genes in the EGTC mouse lines and total genes annotated in MGI. We found the functional distributions for the trapped genes in the EGTC mouse lines and for the RefSeq genes for the whole mouse genome were similar, indicating that the EGTC mouse lines had trapped a wide range of mouse genes.     

Sequence and expression of a novel mouse gene PRDC (protein related to DAN and cerberus) identified by a gene trap approach

Gene trapping in embryonic stem (ES) cells was used to identify a novel gene involved in mouse development. In order to screen trapped ES cell lines for the presence of developmentally regulated genes, an in vitro differentiation test was used. One of the G418 resistant cell lines, in conjunction with the lacZ reporter gene, showed differential expression patterns under differentiated and undifferentiated conditions. The gene trap insertion in this cell line was germ-line transmitted and X-gal staining was used to assess the expression pattern of lacZ in embryos heterozygous for the trapped allele. The reporter gene's expression was detected in commissural neurons in the developing spinal cord, suggesting functions for the trapped gene in mouse neural development. Structural analysis of the cDNA revealed that this trapped gene, named PRDC (protein related to DAN and cerberus), is a novel gene that encodes a putative secretory protein consisting of 168 amino acid residues. PRDC gene product shows limited similarities to the products of DAN (differential screening-selected gene aberrative in neuroblastoma) and cerberus . (DAN is a possible tumor-suppressor for neuroblastoma in human. Cerberus can induce an ectopic head in Xenopus embryos when ectopically expressed.) These three gene products may form a novel family of signaling molecules.     

The identification of new genes: Gene trapping in transgenic mice

Ongoing efforts to clone, sequence and map genes in the mouse have far exceeded our ability to define their functional role. The generation of mutations is an important first step towards understanding the function of genes in normal mouse development and physiology. Gene trapping in embryonic stem cells provides an efficient method to identify, clone and mutate genes at random, permitting the functional analysis of new genes in mice.