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1.
人CD46启动子真核表达载体的构建   总被引:1,自引:1,他引:0  
为了构建人CD46(hCD46)启动子指导目的基因表达的真核表达载体,提取HeLa细胞基因组DNA,用PCR扩增出hCD46基因的启动子区域,序列分析结果表明其与GenBank中hCD46基因5’端某片段的同源性为99.9%。用此启动子替换pcDNA3EGFP中的CMV启动子,并在hCD46启动子和EGFP基因之间插入兔β-球蛋白基因第二内含子(RGI),得到的重组表达载体转染CHO和SP2/0两种鼠源细胞,流式细胞术检测表明CHO细胞EGFP的表达量高于SP2/0细胞,表达特性与人体CD46相似;RGI可以增强EGFP的表达量,但不改变其表达的组织特异性,提示克隆的hCD46启动子可以用于研制模拟人体CD46基因表达特性的转基因小鼠。  相似文献   

2.
胡文革  郝凤霞  陈创夫  王远志  任艳 《遗传》2009,31(10):1029-1036
以开发利用新疆濒危鱼类准噶尔雅罗鱼(Leuciscus merzbacheri)基因资源为研究目的, 利用PCR技术克隆准噶尔雅罗鱼的β-actin 基因, 得到的β-actin 基因片段SZ21包含启动调控区, 大小为2 398 bp。SZ21的启动调控区包括β-actin 基因上游调控序列、第1、第2、第3和第4外显子部分序列。上游调控序列中含有对转录起重要作用的CAAT框、TATA 框和CArG 框等元件。对启动子序列在线分析表明, 获得的启动子含有E-box、RU49、ZBPF、CEBP、CREB等多个重要转录因子结合位点。用AatⅡ破坏真核表达载体pEGFP-N1-AFPⅢ中的CMV启动子, 将准噶尔雅罗鱼的SZ21启动调控区克隆到载体pEGFP-N1-AFPⅢ(CMV坏)上, 构建成重组表达载体β2 pEGFP-N1-AFPⅢ。脂质体转染BHK-21细胞。结果表明, 克隆的准噶尔雅罗鱼β-actin基因启动子SZ21具有启动EGFP报告基因在哺乳动物细胞中表达的活性。通过BHK-21绿色荧光细胞的传代证实, 克隆的启动子具有持续启动蛋白基因表达的活性, 在细胞传代中可以遗传。PCR检测传代的BHK-21绿色荧光细胞基因组DNA, 均能检测到SZ21目的片段。文章成功分离了具有活性功能的准噶尔雅罗鱼β-actin基因启动子。  相似文献   

3.
彭晓  孙燕  刘辉  苟德明  李文鑫 《遗传学报》2004,31(3):221-226
锌指基因家族是人体中最大的基因家族,它参与细胞分化、胚胎发育,并与许多疾病的发生相关。人类ZNF268基因是一个在人胚肝中特异性表达的C2H2型锌指基因,并可能在人的早期肝脏发育中起重要作用。为了研究ZNF268基因表达调控的分子机制,以正常人总基因组为模板PCR扩增了ZNF268基因的5′调控区2533bp片段,并将此片段插入启动子缺失的EGFP(增强型绿色荧光蛋白)载体构建了重组质粒pZNF268—EGFP。用脂质体介导的方法将pZNF268—EGFP转染NIH/3T3、COS7、K562、HeLa4个细胞系。在激光共聚焦显微镜下观察绿色荧光的表达,发现在每个细胞系中,转染了重组质粒pZNF268—EGFP的细胞均有荧光表达,但其起始表达时间均晚于转染了阳性对照质粒pEGFP—C1的细胞且荧光较弱。这表明ZNF268基因的5′调控区2.5kb片段是一个有功能的启动子,但该启动子与CMV启动子相比活性较弱。选择易培养的HeLa细胞系用于缺失研究。将一系列5′端—2456bp至—20bp缺失、3′端均为 77bp的缺失片段插入启动子缺失的CAT(氯酶素酰胺转移酶)载体构建了一系列重组质粒。将这些重组质粒转染HeLa细胞系进行缺失分析,并通过共转染pCMV—Sport—βgal质粒校正转染效率。结果表明,ZNF268启动子—2456~—1639bp区域可能含有正调控元件,—1244~—1013bp和—525~—156bp区域可能含有负调控元件,ZNF268启动子激活转录的一个重要区域位于—156~—20bp。  相似文献   

4.
为了探讨绿色荧光蛋白标记的红色酵母D 氨基酸氧化酶 (DAAO)基因在人宫颈癌细胞 (HeLa细胞 )中的表达及其功能 ,采用基因重组技术构建了含有CMV启动子和EGFP、DAAO基因开放阅读框 (ORF)的真核表达载体 pIRES DAAO。脂质体法转染HeLa细胞 ,荧光显微镜下观察转染细胞中绿色荧光蛋白的表达 ,流式细胞术分析转染效率并筛选荧光阳性细胞 ,命名为HeLa D。以不同浓度的前药D Ala处理HeLa D细胞 ,MTT法检测细胞存活率。结果显示 ,荧光显微镜下可见绿色荧光蛋白在HeLa D细胞中表达 ,流式细胞术成功筛选出HeLa D细胞。前药D Ala能明显杀伤HeLa D细胞。结果表明 ,EGFP可作为报告基因快速筛选DAAO表达载体转染的细胞 ,DAAO/D Ala自杀基因系统可进一步用于肿瘤的基因治疗研究  相似文献   

5.
增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)是一种优化的突变型GFP,DFL是从甘菊中分离出的LFY基因的同源序列。为了研究DFL基因的功能和表达模式,研究利用小片段克隆法将linker序列插入到EGFP基因5′端启始密码子前面,在pBI121载体的CaMV35S启动子的3′端后面插入一段多克隆位点,成功地构建了pBI-DFL-EGFP表达载体。通过设计特异引物,利用PCR技术扩增得了到拟南芥LFY基因的启动子序列,用粘性末端PCR技术将pBI-DFL-EGFP表达载体中CaMV35S启动子替换成LFY基因启动子,构建成了pLFY-DFL-EGFP表达载体。用含有pBI-DFL-EGFP和pLFY-DFL-EGFP质粒的农杆菌侵染洋葱表皮细胞,在荧光显微镜下分别用蓝光激发,均观测到了荧光。这一结果表明,融合蛋白DFL∷EGFP表达载体构建成功,同时还证明了通过PCR技术克隆到的LFY启动子序列具有启动子功能。  相似文献   

6.
用大肠杆菌同源重组获得克隆化重组腺病毒基因组   总被引:8,自引:0,他引:8  
利用大肠杆菌细胞内质粒间同源重组获得克隆化重组腺病毒基因组 DNA,高效构建携带有外源基因的均一重组腺病毒 .将带有狂犬病毒糖蛋白 (GP)基因和加强型 GFP(enhanced GFP,EGFP)表达盒的重组穿梭质粒 p Ad- Track- CMV/ GP与腺病毒骨架载体质粒 p Ad Easy- 1一起同时电击共转化大肠杆菌 BJ51 83.在 BJ51 83细胞内 ,带有同源序列的重组穿梭质粒与骨架载体可进行同源重组 ,得到以质粒形式存在的克隆化重组腺病毒基因组 p Ad- GP’.以 p Ad- GP’为模板 ,经DNA测序确认 GP基因成功整合入此质粒中的腺病毒基因组 E1区外源基因表达盒中 .线形化的p Ad- GP’转染 2 93细胞后可得到基因组结构均一、在 E1区插入有 GP和 EGFP表达盒的重组腺病毒 ,病毒滴度可达 1× 1 0 8pfu/ ml.电镜下此重组病毒颗粒直径约为 70 nm,略呈球形 ,用荧光显微镜观察感染细胞有很强的 EGFP表达 .实验表明 :利用大肠杆菌同源重组获得克隆化的重组腺病毒基因组 DNA,可高效制备高滴度的均一重组腺病毒  相似文献   

7.
粘虫β-actin基因cDNA的克隆、序列分析及表达量检测   总被引:1,自引:0,他引:1  
李柯  阴环  奚耕思  廉振民 《昆虫知识》2010,47(6):1089-1094
β-actin基因作为actin家族的一员,在基因定量实验中常用作内参基因。本实验运用RT-PCR和RACE技术,以粘虫Mythimna separata(Walker)cDNA为模板,对β-actin基因进行克隆获得全长cDNA序列,并利用生物信息学方法,对β-actin基因全长cDNA序列及推测得到的β-actin蛋白序列进行分析。结果表明,获得的粘虫核β-actin基因cDNA序列长度为1 472 bp,其中包括68 bp的5′非编码区、273 bp的3′非编码区和1 131 bp的开放阅读框,编码一个376个氨基酸蛋白,具有actin蛋白家族典型特征。推测得到的粘虫β-actin蛋白理论分子量为41.7497 ku,等电点为5.29,富含6种类型的特定功能位点。该蛋白序列与其他动物β-actin蛋白序列具有97.9%~99.7%高度同源性。β-actin表达量检测结果显示β-actin在6种不同组织间表达无显著差异(P>0.05),表明β-actin可作为研究粘虫不同基因表达水平高低的可靠内参基因。该基因的cDNA序列已经递交GenBank并获得登录号为GQ856238。  相似文献   

8.
根据已发表的β-酪蛋白基因全序列设计合成引物,以西农萨能奶山羊乳腺组织基因组DNA为模板,通过长链PCR扩增β酪蛋白基因组基因5’侧序列。将该序列克隆入pMD18-T载体,并对其进行序列测定。结果克隆得到西农萨能奶山羊β-酪蛋白基因-4 359至+2 106共6465bp序列。该序列保留β-酪蛋白第一内含子、第一外显子和第二外显子及其信号肽部分。将该序列与已公布的其他山羊β-酪蛋白基因序列进行比较,其同源性为96%-98%。生物信息学分析显示,β-酪蛋白基因启动子区包含有TATA启动子序列和HOXF、Oct-1、AP1、CEBP、STAT、NFKB、GR、ER、PR和ETS等转录因子识别位点。将此6 465bp片段插入荧光素酶报道基因载体pGL3-Basic中,构建重组质粒pGL3/B65,瞬时转染萨能奶山羊原代培养的乳腺上皮细胞48h后检测显示,该假设的启动子区确有启动子功能,对泌乳激素发生反应。本研究为进一步构建有效的乳腺生物反应器载体奠定了基础。  相似文献   

9.
目的克隆扩增APC11开放阅读框基因片段,构建其真核表达重组质粒,为后续研究其功能做准备。方法按照GenBank中人APC11序列设计引物,以HeLa细胞cDNA为模板,扩增出基因片段。该片段与真核表达载体pEGFP-C1连接,得到的重组质粒经双酶切和测序鉴定后用Western印迹检验表达。结果扩增片段长度为285bp,重组真核表达质粒经双酶切鉴定后测序鉴定正确。Western印迹证实pEGFP—C1-APC11在真核细胞内表达。结论成功克隆了APC11基因并构建了pEGFP—C1-APC11真核表达重组质粒,Western印迹证实其表达良好,对该基因功能的深入研究打下了坚实基础。  相似文献   

10.
提取马立克氏病毒Ⅰ型疫苗毒株CVI988的总DNA为模板,利用PCR技术扩增出病毒生长非必需的US2基因并克隆入T—easy载体。将CMV启动子和增强子控制的含GFP基因表达盒克隆入US2基因中,成功构建了含GFP基因的转移质粒载体pGUS2GFP。用脂质体将其与CVI988株共转染CEF细胞,用96孔板稀释法得到纯化的表达绿色荧光蛋白的重组CVI988病毒株rCVIGFP,并分别测定其在体内和体外的生长情况。表达EGFP基因的重组病毒在细胞上生长曲线与亲本毒CVI988类似,体外实验表明,1日龄腹腔接种该重组毒后,可以从鸡体内分离到表达绿色荧光的病毒。  相似文献   

11.
A 3 338 bp DNA fragment including the open reading frame and 5′-flanking region of β-actin gene for black carp genome was obtained through PCR amplification. Analysis of the sequencing results indicated the ORF of black carp β-actin gene encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The black carp β-actin sequence showed 100% identity to common carp, grass carp, and zebrafish, 99.2% identity to human and Norway rat β-actin gene, 98.9% and 98.1% identity to chicken and Kenyan clawed frog β-actin gene, respectively. The promoter region of black carp β-actin gene was inserted into the promoterless pEGFP1 vector. The recombinant plasmid was microinjected into the fertilized eggs of mud loach before two-cell stage as well as transfected into HeLa cell line. GFP expression was found in 50% of mud loach embryos and 2/3 HeLa cells. The GFP expression could be observed in every part of the mud loach embryos, and in some embryos, the GFP was expressed in the whole body. Thus, the usefulness of black carp β-actin promoter as a ubiquitous expression promoter was confirmed using the EGFP as a reporter gene.  相似文献   

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13.
An effective foreign gene transfer method for shrimp would have several potential uses in the shrimp culture industry, such as in preventing infectious diseases. We evaluated two gene transfer methods and used black tiger shrimp, Penaeus monodon, as a model target species. For a promoter, we used the 1,592-bp promoter region of the EF-1alpha gene, a house-keeping gene, of kuruma shrimp Marsupenaeus japonicus. The promoter region was linked to either the gene for green fluorescence protein (GFP) or the gene for chloramphenicol acetyl transferase (CAT). The fusion genes were designated pJEF-GFP and pJEF-CAT, respectively. The pJEF-GFP gene was introduced into fertilized eggs of black tiger shrimp by microinjection and particle gun bombardment. The survival rate of the microinjected eggs was 17.6%, and 1.0% of the treated embryos were found to be GFP-positive. However, the GFP-positive embryos were damaged and embryogenesis did not progress. The survival rate of the particle-bombarded eggs was 60.6%, and 0.42% of the treated embryos were found to be GFP-positive. Ubiquitous GFP expression was observed from 8 hr post-fertilization and these embryos developed and hatched normally. The pJEF-CAT gene was introduced into fertilized eggs of black tiger shrimp using the optimized conditions of the particle gun bombardment. CAT activity was observed from 1 to 7 days post-fertilization, with the highest activities being observed at 5 and 7 days post-hatching.  相似文献   

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15.
We have analysed the correlation between expression and methylation for the human β-actin promoter introduced into mouse embryos. The β-actin promoter was fused to the reporter gene lacZ, and expression was analysed after pronuclear injection into fertilized mouse eggs. We analysed transient expression in in vitro cultured preimplantation embryos and expression after chromosomal integration in 5 independent lines of transgenic mice. The in vitro cultured preimplantation embryos expressed lacZ from the 2-cell to the blastocyst stages, and most abundantly at the morula stage. By increasing the amount of injected DNA, a larger proportion of embryos expressed lacZ. Embryos expressing lacZ in only a subset of the blastomeres were detected at all preimplantation stages. In contrast to the transient expression after injection, we have not detected lacZ expression in any of the 5 analysed lines of transgenic mice carrying the same construct. The lack of expression in transgenic mice correlates with hypermethylation of C residues in the vast majority of CG sequences in the integrated β-actin/lacZ construct, whereas the injected construct was completely nonmethylated. We discuss methylation and other possible reasons for the observed differences in expression between injected and integrated copies of the β-actin/lacZ construct and for lacZ expression in only a subset of blastomeres in preimplantation embryos. © 1993 Wiley-Liss, Inc.  相似文献   

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两种不同终止子在转基因鲤鱼中的促生长效应   总被引:2,自引:2,他引:0       下载免费PDF全文
钟山  罗大极  吴刚  徐婧  汪亚平  朱作言 《遗传》2009,31(8):831-836
转基因构建体中启动子的选择会直接影响转植基因的活性, 近年来有研究表明转基因构建体中终止子的选择会一定程度地影响转植基因的活性。为了更好地筛选转基因构建体和培育快速生长的转“全鱼”生长激素(Growth hormone, GH)基因鱼, 文章用鲤鱼β-actin基因终止子和生长激素基因终止子分别构建了转基因构建体, 显微注射得到转“全鱼”GH基因鱼P0代养殖群体, 比较两种不同终止子构建体的活性。统计分析发现, 生长激素基因终止子构建体的养殖群体的体重频率呈正态分布且平均体重显著高于β-actin基因终止子构建体的养殖群体, β-actin基因终止子构建体的养殖群体的体重频率呈右倾趋势的非正态分布。值得一提的是在混合养殖组中得到一条生长最为快速的鲤鱼证实为转基因阳性且为生长激素基因终止子构建体的转基因鲤鱼。该结果表明转“全鱼”生长激素基因鲤鱼可快速生长, 并能将转植基因向下代遗传。实验结果提示生长激素基因终止子构建体比β-actin基因终止子构建体表现的促生长活性要强。  相似文献   

18.
Two full-length cDNAs encoding glutathione S-transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 +/- 6.6 (grass carp), 103.1 +/- 8.9 (bighead carp), 92.6 +/- 15.0 (crucian carp), 72.3 +/- 7.8 (mud carp), 58.8 +/- 11.5 (silver carp), and 33.6 +/- 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed.  相似文献   

19.
转基因红鲤体细胞的核移植   总被引:2,自引:0,他引:2  
赵浩斌  朱作言 《遗传学报》2002,29(5):406-412
以F4代转hGH基因红鲤体细胞(肾脏和尾鳍)及培养18代的F4代转hGH基因红鲤尾鳍细胞为核供体,泥鳅或黄河鲤成熟卵为受体,进行了核移植,以探讨外源F4代转基因鱼体外源基因的分布与存在形式,稳定性和克隆转基因鱼的可能性。F4代红鲁肾脏细胞核与泥鳅卵配合的核移植胚胎有12.4%发育到囊胚,0.33%发育到神经胚;F4代尾鳍细胞核移入泥鳅卵后的重组胚发育到囊胚,神经胚、肌节期和肌肉效应期的胚胎分别为24.5%、0.3%、0.2%和0.1%;对照卵无发育。F4代红鲤尾鳍培养细胞与黄河鲤卵子配合的重组胚胎有50.53%发育到囊胚,5.69%发育到原肠胚,0.53%发育到神经胚,0.4%发育到肌节期。说明由于同种细胞核与卵细胞的相容性高于异种核卵的相容性,早期发育率高;而由于培养细胞的异倍化,后期的发育率降低。用PCR技术对供体鱼不同个体及同一体不同组织外源基因检测,结果100%个体为阳性鱼,而且不同组织的阳性率也是100%,说明外源基因均匀分布在不同组织中。无论F4代转基因鱼的肾脏细胞、尾鳍细胞还是培养的尾鳍细胞作核移植供体,核移植胚胎中hGH基因的检出率为100%。说明F4代转基因红鲤个体不同细胞都存在hGH基因,而且经长期培养不会丢失。表明F4代转基因红鲤中的外源hGH基因已基本稳定,体细胞核移植可以作为获得同质化转基因鱼的有效手段,但核移植效率还很低。另外还讨论了核质的相容性、细胞周期的协调、染色体的变异等因素对核移植的影响。  相似文献   

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