目的 利用CRISPR/Cas9技术,获得环指蛋白126(RNF126)基因敲除小鼠模型.方法 针对C57BL/6小鼠Rnf126基因的第四外显子设计敲除引物,构建sgRNA重组表达载体.通过体外转录获得sgRNA及Cas9 mRNA,并以显微注射的方式将RNA导入到C57BL/6小鼠的受精卵中,通过胚胎移植至代孕母鼠.获得子代工程鼠后,用PCR对其进行鉴定.结果 成功获得针对鼠Rnf126基因的sgRNA以及Cas9 mRNA;通过显微注射获得了82枚状态良好的受精卵并成功移植至3只代孕母鼠体内;获得了11只F0代仔鼠,鉴定后选择一只单链缺失62个碱基的阳性鼠进行扩繁并在F1、F2代检测到该突变.结论 成功构建Rnf126基因敲除C57BL/6小鼠,该模型可用于RNF126基因及其表达产物的生物学功能研究.

Objective To establish a method for RT-PCR detection of mouse hepatitis virus (MHV) and provide a rapid method for monitoring the daily production process of mice. Methods The total RNA form mouse colon tissue was extracted. The first strand c DNA was synthesized by reverse transcription reaction. Then using the c DNA as the template, a DNA fragment of MHV was amplified by PCR with four pairs of specific primers. The PCR reaction conditions (including primers, primer concentration, annealing temperature, template concentration, etc.) were optimized. Results The two pairs of MHV primers were effective and specific. The PCR results of these two pairs of primers were consistent with the results of ELISA. Conclusion The RT-PCR detection method of MHV was specific and relatively simple. It can be used as a rapid method for MHV detection in the production process of mice.

目的 利用CRISPR/Cas9慢病毒载体系统建立小鼠原代卵巢上皮细胞TP53基因稳定敲除细胞系,分析细胞增殖、细胞周期、克隆形成以及细胞转移侵袭能力的变化.方法 构建LentiCRISPRv2-sgRNA TP53基因敲除质粒,用293FT细胞进行慢病毒包装,转导小鼠原代卵巢上皮细胞,嘌呤霉素筛选出稳定敲除细胞系,进行PCR、蛋白免疫印迹以及免疫荧光鉴定.细胞增殖、细胞周期变化、克隆形成、细胞迁移侵袭能力分别用MTT、流式细胞分析、单层培养以及Transwell小室进行测定.结果 TP53基因敲除小鼠原代卵巢上皮细胞中P53表达缺失;TP53敲除引起细胞迅速增殖,DNA合成加速,克隆形成以及迁移侵袭能力增强.结论 获得了原代小鼠卵巢上皮细胞TP53基因稳定敲除细胞系,细胞生物学特征明显改变.

Objective BALB/c mutant curly mice and normal BALB/c mice were genetically detected by microsatellite DNA marker analysis to detect the differential microsatellite loci between BALB/c mutant curly mice and normal mice.Methods 38 microsatellite DNA loci were selected and their variation in the BALB/c mutant curly mice, BALB/c mutant hairless mice and normal BALB/c mice were detected by multiplex fluorescence PCR and STR scanning genotyping.Results There were 27 the same microsatellite loci between the 38 microsatellite loci in BALB/c mutant curly mice and normal mice,and there were 11 differential loci, with a mutation rate of 28.9%(11/38). There were 30 the same sites between BABL/c mutant hairless mice and normal mice,and there were 8 different loci,with a mutation rate of 21.1%(8/38). There were also 12 differential loci between BABL/c mutant curly mice and hairless mice. Conclusions BALB/c mutant curly mice have a higher mutation rate and are significantly higher than those of hairless mice,demonstrating that the mutations in curly mice and hairless mice are two completely different mutations. These results provide reliable theoretical data for the future study and development of BALB/c mutant curly mice.

目的 获得自然杀伤(NK)细胞缺陷的小鼠动物模型,并研究溶酶体运输调节因子(Lyst)基因的功能.方法 采用CRISPR/Cas9技术敲除C57BL/6小鼠的Lyst基因:针对小鼠Lyst基因编码区第50个外显子,设计CRISPR/Cas9靶点引物(sgRNA),并重组构建pUC57-Lyst-sgRNA载体.将重组质粒体与Cas9质粒分别利用T7 RNA聚合酶体外转录为mRNA,并按比例显微注射入小鼠受精卵,将受精卵移植到受体动物.获得子代小鼠后,利用PCR扩增和测序方法筛选Lyst基因发生改变的动物个体.结果 显微注射移植后获得7只F0代基因敲除小鼠,且毛色均发生了明显的变化.结论 利用CRISPR/Cas9技术可以构建Lyst基因缺陷小鼠,外观表型与已有的文献报道一致.

为了比较Wnt通路重要结合受体Frizzled家族的Frizzled 3、Frizzled 4受体蛋白基因在黑线仓鼠及其白化突变系(A:CHA)皮肤组织的表达差异,分析这两种基因对白化性状产生的影响,试验提取黑线仓鼠和A:CHA的皮肤组织总RNA,利用普通PCR方法筛选引物,通过实时荧光定量PCR技术比较黑线仓鼠和A:CHA皮肤组织Frizzled 3、Frizzled 4基因表达量的差异.结果表明:黑线仓鼠皮肤中的Frizzled 3、Frizzled 4基因mRNA的相对表达量分别是A:CHA组织中的4倍、5倍.Frizzled 3、Frizzled 4基因在A:CHA皮肤组织中的表达量下调.说明Frizzled 3、Frizzled 4基因与A:CHA白化性状的产生存在一定关联.

Objective To knockout the MATP gene of mouse melanoma cell line B16F10 using CRISPR/Cas9 system,and to lay foundation for the functional study of MATP gene.Methods Specific primers of MATP were designed according to the report in http://crispr.mit.edu/ website.The primers were linked to pCAS9/gRNA1 vector.Then the positive vector was transfected into mouse melanoma B16F10 cells,and monoclonal cell lines were obtained by the infinite dilution method.After the genomes of different monoclonal cell lines were extracted and sequenced,the cell lines with MATP gene cleavage were screened,and the expression of MATP in these cell lines was verified by Western-blot analysis.Results Three MATP gene knockout cell lines were successfully obtained.The western-blot results showed that the cell lines did not express MATP protein.Conclusions The knockout of MATP gene in B16F10 cell line can be successfully achieved using the pCAS9/gRNA1 vector.

Objective:To explore whether exosomes originated from tumor cells included parent cells-derived fusion gene mRNA.Methods:Firstly,exosomes were extracted from the supematant ofH3122 cells by the ExoQuick kit and western blot analysis was adopted to verify that exosomes were successfully extracted.Secondly,Total RNA was extracted from both the cell lysate and exosomes of H3122 cells and reverse transcription was performed.Subsequently,qualitative PCR of cDNA was performed with the primer used to amplify EML4-ALK variant 1.Finally,the PCR products were sent to be sequenced and we blasted and analyzed the sequencing results.Results:We successfully exacted exosomes produced from the H3122 cells,and detected EML4-ALK mRNA in the exosomes from the H3122 cells.The sequencing results showed that both the H3122 cells and the exosomes derived from the H3122 cells harbored EML4-ALK fusion variant 1,which also demonstrated that we could detect the mRNA or gene fusion of the tumor cells through the exosomes from the tumor cells.Conclusion:Exosomes secreted by tumor cells not only contain parent cells-derived mRNA but also reflect some biological characteristics including gene fusion of the parent cells.

通过构建比格犬ERβ稳定细胞系及其配体结合区(LBD)的真核表达载体,为探讨ERp的功能奠定基础.以重组质粒pEGFG-N 1-ERβ为模板,扩增出全长ERβ以及ERβ-LBD基因片段,经胶回收纯化后连接到pMD18-T载体,经测序鉴定后再连接至Lenti-OE-Flag以及pFLAG-CMV4载体.使用HEK-293T包装细胞系,包装Lenti-OE-Flag-ERβ产生慢病毒,并将所得病毒悬液感染HEK293细胞,利用Puromycin加以筛选,获得稳定表达细胞系.同时将pFLAG-CMV4 ERβ-1BD质粒瞬时转染293T细胞,采用Western blot方法验证ERβ以及ERβ-LBD蛋白的表达情况.结果表明,试验成功获得ERβ稳定表达细胞系和重组质粒pFLAG-CMV4-ERp LBD;Western blot方法证实,稳定表达细胞系可以成功表达ERβ蛋白,ERβ-LBD也可以瞬时成功表达.

目的 探讨大肠杆菌O127∶H6 (Escherichia coli O127∶H6)感染BALB/c小鼠建立肠炎模型及其受激活调节正常T细胞表达和分泌活性因子(RANTES)与炎症反应的相关性.方法 通过不同剂量大肠杆菌O127∶H6灌胃的方式,建立肠炎小鼠模型(实验组),灌胃生理盐水为对照组.取小鼠空肠标本,石蜡包埋,HE染色,观察病理变化;采用荧光定量PCR的方法,测定不同剂量组感染小鼠空肠组织的RANTES表达情况.结果 成功建立了肠炎性小鼠模型,实验组与对照组比较,空肠有明显的炎性病理变化;荧光定量PCR结果表明实验组RANTES表达量明显高于对照组.结论 通过灌胃大肠杆菌O127∶H6的方式可以建立肠炎小鼠模型,其空肠部位具有明显的病理变化;RANTES表达量的变化可以作为评价小鼠肠炎的指标之一.

目的 构建慢病毒介导的NogginRNAi干扰序列,并分析这些干扰序列对Noggin基因的沉默效果.方法 针对目的基因Noggin的mRNA设计四条干扰序列,并将这些序列连接到Lenti-KD慢病毒载体,将重组质粒瞬时转染HEK-293T包装细胞,获得重组慢病毒.将重组病毒感染MC3T3-EI细胞,利用puromycin进行筛选,获得稳定表达细胞系.通过实时荧光定量PCR和Western blot技术分析不同干扰序列的干扰效果.结果 实时荧光定量PCR结果显示,四种干扰序列对Noggin基因的表达都有一定的沉默效果,但只有shNoggin-1(P＜0.01)对其表达影响显著.Western blot结果显示,四种干扰序列中只有shNoggin-1 (P ＜0.01)对Noggin的表达蛋白具有显著的降低作用.结论 获得了一种Noggin基因的干扰序列,该序列能够干扰Noggin基因mRNA的稳定性,从而影响蛋白的表达.该干扰序列可以用于部分敲除Noggin基因,从而用于研究Noggin基因的功能.

为了建立ERβ1293的稳定表达细胞系,为ERβ1293的功能研究奠定基础.以重组质粒pEGFG-N1-ERβ1 293为模板,扩增出全长ERβ1293,经胶回收纯化后连接到pMD18-T Vector,经测序鉴定后双酶切连接至Lenti-OE-Flag载体,重组质粒经PCR、双酶切、测序鉴定;使用HEK-293T包装细胞系,包装产生慢病毒.将所得病毒悬液感染HEK293细胞,利用Puromycin加以筛选,获得稳定表达细胞系,并采用Western blot方法验证基因表达情况.结果获得了Lenti-OE-Flag-ERβ1293重组质粒,并成功包装成慢病毒.利用病毒感染HEK293细胞后获得了稳定表达细胞系,Westernblot方法证实,稳定表达细胞系可以成功表达ERβ1293蛋白,为研究ERβ1293的功能奠定了基础.

Objective To analyze the effect of Noggin silencing on the BMP and Wnt signaling pathways in hair follicle development.Methods The expression of BMP-2, BMP-4, BMPR-IA, BMP-6, BMP-7, LEF-1 andβ-catenin in Noggin silencing MC3T3-E1 stable cell line was detected by RT-PCR and western blot.Results RT-PCR results showed that the expressions of five genes in BMP signaling pathway were all significantly influenced by Noggin silencing, the ex-pressions of BMP-2 (P<0.001), BMP-4 (P<0.01), BMP-6 (P<0.001) and BMP-7 (P<0.001) were all increased and the expression of BMPR-IA (P<0.01) was decreased.While the expressions of the two genes LEF-1 (P<0.001) and β-catenin ( P<0.001) in Wnt signaling pathway were significantly decreased.Western blot results showed that the ex-pressions of these proteins in the two signaling pathways were also affected.The expressions of BMP-2 (P<0.05), BMP-4 (P<0.05), BMP-6 (P<0.05) and BMP-7 (P<0.05) were all increased, while the expressions of BMPR-IA (P<0.05), LEF-1 (P<0.01) andβ-catenin (P<0.001) were decreased.Conclusions There may be a negative feedback regulation of Noggin on the BMP signaling pathway in vitro, but a positive feedback regulation on the Wnt signaling pathway in vitro.It provides certain evidence for studies on the effect of Noggin gene on BMP and Wnt signaling pathways in vivo. There may be an interaction between hair follicle development-related signaling pathways, which still needs further experi-ments to prove.

Objective To establish a stable transfection cell line of iRhom2 and its mutant through recombinant lentivirus infection.Methods The full-length gene of iRhom2 and its mutant were cloned into the lentivirus vector Lenti-OE-Flag, and got recombinant lentiviral vector of Lenti-OE-iRhom2 and Lenti-OE-iRhom2mut.The constructed recombi-nant lentivirus vectors were transfected into HEK-293T packaging cells to obtain the recombinant virus.Vero cells were in-fected with recombinant virus.The stable expressing cell lines were obtained by pressure screening with puromycin. Results The recombinant lentivirus vectors were constructed and the recombinant virus was obtained.The stable express-ing cell lines were obtained using virus infection and the protein expression was testified with Western blotting.Conclu-sions Stable iRhom2-expressing Vero cell line and its mutant are achieved by recombinant lentivirus infection.It paves the way for future study on biological functions and mechanism of iRhom2.

Objective To establish an eukaryotic vector of monkey B virus glycoprotein D gene and analyze the expression of gD gene in human embryonic kidney 293T cells.Method First, the protein of monkey B virus glycoprotein D was obtained by gene synthesis.The gene fragments were digested with Pst I and Not I, and ligated to pEGPF-N3. Then, the recombinant plasmid pEGPF-N3-GD was transfected into 293T cells.The expression of gD protein in the cells was detected by Western blot, and the expression localization was investigated using laser scanning confocal microscopy. Results The recombinant plasmid pEGPF-N3 carrying gD gene was successfully constructed, and normally expressed in the 293T cells.Conclusions Glycoprotein D of monkey B virus is expressed successfully in the 293T cells and the protein is located on the cell surface.It may be useful for the preparation of specific recombinant antigen to the glycoprotein D of monkey B virus on cell surface, and can be also used for preparation of antigen slide for detection of monkey B virus.

Objective To screen the splicing isoforms of estrogen receptor βin the Beagle hypothalamic -pituitary -gonadal axis.Methods For ERβmRNA CDS sequence of eight exons, primers were designed confined to the CDS sequences of two sequential exons.Beagle hypothalamus, pituitary, ovary and uterus tissue cDNA were used as template, and corresponding sequences were amplified by PCR.PCR products were sequenced and aligned in the NCBI web site.The correct gene was then analyzed with DNAMAN comparative analysis software and handwork checking up, thus got the ERβsplicing isoforms of Beagle. Results Four beagle ER beta splicing isomers were obtained:exon 4 complete skipping ER βisomer (300 bp missing), two kind of Beagle ERβisoforms with partial exon 4 and partial exon 5 complicated missing (isoformⅠ334 bp missing and isoformⅡ265 bp missing), and exon 7 complete missing ERβsplicing isoforms (181 bp missing).Exon 4 complete skipping and exon 7 complete missing isomers had been obtained full length coding sequence, and the other two splicing isomers were partial coding sequence.Conclusion This project gained four ERβsplicing isomers of Beagle, and that will lay an important foundation for further study of their roles in the Beagle reproductive regulation mechanism.

Objective To get the gene encoding extracellular domains of gB protein of B virus and analyze its expression in the eukaryocyte cell.Methods synthesizing gene fragment encoding extracellular domains of gB protein of B virus was by using synthesis gene, then digested with the restriction endonucleases BamHⅠand NotⅠand inserted into eukaryotic expressing vector pEGFP-N3.pEGFP-N3-GB合 was transfected into 293 cells.After protein extraction, the expression of gene was detcted by western blotting, and the cellular localization of the gene was analyzed by immunofluorescence and laser scanning confocal microscopy.Results pEGFP-N3-GB合were expressed in 293 cells and on the cell membrane.Conclusion eukaryotic expressing system can produce specific antigen recombination protein of B virus gB protein and express on the cell membrane.

Objective To clone and sequence the dystonin variant X1 gene of Cricetulusbarabensis and the albino mutant Cricetulusbarabensis so as to find out the difference of encoding arear of the muscular ribosome between Cricetulusbarabensis and the albino mutant Cricetulusbarabensis.Methods According to the same type of abnormal muscle tone protein of the mice and rats, we designed 6 pairs of primers, and got their cDNA genes from skins of the Cricetulusbarabensis and the albino mutant Cricetulusbarabensis by RT-PCR amplification, then cloned and sequenced. Results Sequence alignment showed 17 variances in coding areas, and 24 in amino acid, but no in key nucleic acid and protein.Conclusion The variances in coding areas will not lead to the albino, and its mechanism requires further investigation.

Objective To construct recombinant eukaryotic expression vectors of Beagles ERβ1293,and identify the expression and localization of ERβ1293 in the human embryonic renal HEK293T cells by Western blotting and indirect immunofluorecent assay. Methods The ERβ1293 gene was amplified by PCR using pEGFP-N1-ERβ1293 as a template. The recombinant eukaryotic expression vector of pcDNA3. 1-Myc-ERβ1293 was constructed,and transfected it into HEK293T cells. The expression of pcDNA3. 1-Myc-ERβ1293 was detected by Western blot,and its localization was detected by indirect immunofluorecence assay. Results The expression of pcDNA3. 1-Myc-ERβ1293 was constructed, and successfullyidentified in the HEK293T cells using Western blotting,and detected only in the cytoplasm using IF by laser scanning confocal microscopy. Conclusions In the previous experiment,the coding sequence of Beagles ERβ1593 splice variants has been obtained,which is lack of exon 4,and its ligand binding ability is reduced or disappeared,so that the intracellular localization of ERβ1293 codeing protein is changed.