Application of a nuclear localization signal gene in transgene mice

Efficient gene transfer by cytoplasm co-injection will offer a powerful means for transgenic animals. Using co-injection in cytoplasm, two independent gene constructs, including bovine α-s1-casein-hG-CSF and a mammal expression vector expressing a nuclear localization signal (mNLS), were introduced into fertilized mouse eggs. The target gene construct was docked into host nucleus probably by the nuclear localization signal. Transgene mice have been obtained at 58% (29/50) of integration ratio. Expression level of the positive transgene mice was detected by Western blotting. Maximal expression of human G-CSF was estimated about 540 mg/L of milk. The expression ratio was up to 75% (9/12). The results here have important practical implications for the generation of mammary gland bioreactors and other transgene studies. Co-injection of a target gene with an expression vector of a mammal nuclear localization signal by cytoplasm appears to be a useful, efficient and easy strategy for generating transgenic animals, which may be able to substitute the routine method of pronucleus-injection of fertilized eggs.     

High expression of human serum albumin in milk of trans- genie mice directed by the goat p-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene pro-moter, 5'upstream regulatory region, exons 1, 2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F0 mice were obtained. Of the 33, 8 mice (5 , 3 ) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.     

High expression of human serum albumin in milk of transgenic mice directed by the goat β-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene promoter, 5′upstream regulatory region, exons 1,2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F₀ mice were obtained. Of the 33, 8 mice (5♀, 3 ♂) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.     

Pygo2转基因小鼠模型的建立及表型的初步分析

主要通过建立Pygo2转基因小鼠的模型对其表型进行初步分析.首先构建K14-2×flag-Pygo的转基因构件,经酶切、纯化后构建Pygo2转基因小鼠.出生后的仔鼠用PCR和Western方法检测基因型,并通过进一步的免疫组化验证Pygo2基因的表达.PCR检测获得7只转基因阳性鼠,6只Western检测为阳性.对转基因小鼠子代的胚胎和成体进行免疫组化证明,Pygo2基因在皮肤和乳腺组织中有过量表达.转基因小鼠的皮肤、乳腺以及鼠尾椎骨等组织出现了异常的表型.乳腺中有肿瘤组织的形成,且Pygo2在肿瘤中有大量表达.该模型的成功建立为进一步研究Pygo2的功能奠定了基础.     

人白介素-3乳腺表达载体的构建

人白介素-3是一种造血系统和免疫系统调节剂,在治疗造血系统疾病、肿瘤、先天或获得性免疫功能缺陷等方面发挥着重要作用.应用牛乳腺生物反应器生产人白介素-3的研究具有重要的临床应用和经济价值.研究选用具有红色荧光蛋白报告基因(R ed2)和新霉素抗性基因(neor)表达框架的pD sR ed2-1质粒为骨架构建人白介素-3乳腺表达载体.通过PCR方法分别扩增牛β-酪蛋白基因5′端上游调控序列、人IL-3基因以及CM V启动子序列,将它们按先后顺序分别定向克隆于质粒pD sR ed2-1的多克隆位点内,使牛β-酪蛋白基因调控序列位于人IL-3基因的上游,指导人IL-3基因在乳腺组织中特异性表达,而CM V启动子位于红荧光蛋白基因的上游,指导红荧光蛋白基因在所有的组织中非特异性表达.限制性酶切片段分析及部分DNA序列鉴定结果表明,所构建载体结构正确.     

Site-independent expression of the chicken beta A-globin gene in transgenic mice

The level of expression of exogenous genes carried by transgenic mice typically varies from mouse to mouse and can be quite low. This behaviour is attributed to the influence of the mouse chromatin near the site of transgene integration. This 'position effect' has been seen in transgenic mice carrying the human beta-globin gene. It was however, abolished when DNase I hypersensitive sites (normally found 65 to 44 kilobases (kb) upstream) were linked to the human beta-globin transgene. Thus, the upstream DNA (previously named a dominant control or locus activation region, now denoted a locus control region) conferred the ability to express human beta-globin at high levels dependent on copy number on every mouse carrying the construct. We report here an investigation of chicken beta A-globin gene expression in transgenic mice. A 4.5-kb fragment carrying the beta A-globin gene and its downstream enhancer, without any far upstream elements, is sufficient to ensure that every transgenic mouse expresses chicken globin messenger RNA at levels proportional to the transgene copy number. Thus the chicken DNA elements that allow position-independent expression can function in mice. In marked contrast to the human beta cluster, these elements are no farther than 2 kb from the gene. The location of the elements within the cluster demonstrates that position independence can be mediated by DNA that does not define a gene cluster boundary.     

Expression of recombinant human lysozyme in the milk of transgenic mice

Human lysozyme is a 130-aa (amino acid) alkaline polypeptide, and has both anti-bacterial and anti-viral properties which make it an important component of human natural immunity system. As a first step toward the ultimate goal of improving the anti-bacterial properties of bovine and ovine milk, a transgenic mouse that contains the genomic DNA sequence of the human lysozme gene has been generated for the first time. From 83 mice generated by microinjection, a total of 6 positive transgenic mice were identified by PCR and Southern blot. F1 mice positive for transgene in lines were also detected by PCR. This shows that transgene could be transmitted from founder transgenic mice to their offspring. Recombinant human lysozyme (rHlys) was found in the whey of 3 female positive transgenic mice by Western blot. The highest concentration of rHlys for transgenic mice was 0.2mg/mL. The antibacterial activity of the whey for transgenic mice was highly enhanced up to 0.4 times as much as that of human, while that of non-transgenic mouse was very low. Although the lysozyme activity of transgenic mice is still lower than that of human, the rHlys exhibits the same specific activity as that of human lysozyme. It provides a strong basis for further studies into the possible application of rHlys express in mammary gland.     

外源基因在转基因动物乳腺中的特异性表达研究

动物基因工程研究最大的突破就是转基因动物研究的进展,自八十年代初第一批转基因小鼠问世以来,转基因动物的研究已从方法学研究步入了应用性研究阶段,转基因动物除作为研究工具广泛应用于发育生物学、免疫学、遗传学以及医学等生命科学领域外,外源基因在转基因动物的特异性表达,尤其是在乳腺的表达,又可将转基因用作生物反应器进行了生物活性蛋白的生产而于商业生产,在转基因动物乳腺的特异性表达研究中,寻找乳蛋白基因调控     

Building of hFⅨ transgenic mice by spermatogenic cells

Human FⅨ expression vector pCMVⅨ was packaged by effectene^TM reagent and injected into mice seminiferous tubules with glass pipettes.The expressional frame of pCMVⅨ was examined by PCR and Southern blotting among 41 progenies.There were 2(4%) mice being integrated with hFⅨ gene into chromosomes.4.6ng/mL of hFⅨ protein was expressed in plasma of one mouse,which was tested by ELISA.We demonstrated that building of transgenic animals by spermatogonial stem cells is an efficient method.Meanwhile,it has also been proved to be an alternative choice for mammary gland bioreactor.     

Vill转基因小鼠的制备

摘要:目的　通过制备Vill转基因小鼠研究该基因的功能。 方法与结果　首先由RT-PRC方法获得全长约2605bp的Vill基因;经T载体克隆测序验证后,以克隆载体pMD19-T-Vill为模板,设计引物并引入酶切位点,将PRC扩增产物与pEF6/V5-His-LacZ同时进行酶切、连接,构建表达载体pEF6/V5His-Vill;经真核表达验证后,酶切获得含Vill基因的显微注射DNA构件;显微注射390枚受精卵后,在出生存活的77只仔鼠中获得转基因阳性GO代小鼠19只,其中16只能够稳定遗传并建系,转基因阳性小鼠外观未有明显改变。 结论　Vill转基因小鼠为该基因的功能研究准备了实验材料。     

基因表达系统与转基因动物乳腺反应器

出于研究、医疗或工业目的，常常需要大量置备各种生物活性蛋白质，为此已经建立了多种基因工程表达系统，如微生物发酵系统、真核细胞培养系统、转基因植物表达系统和转基因动物表达系统等。近几年，利用转基因动物作为生物反应器由乳汁中生产蛋白药物的研究取得了很大进展，有多种动物可被用于转基因。基本过程是将基因构件显微注射到单细胞期受精卵，基因构件以一定几率整合到受体基因组中。通常转基因和其表达模式可忠实地遗传。许多蛋白将以高浓度低成本由转基因家畜的奶中生产。这些转基因动物与传统的动物细胞培养和细菌发酵技术相比尤显高效。乳腺能完成包括二硫桥形成、酰胺化、羧基化和糖基化在内的翻译后修饰，１头转基因羊就是１套发酵罐。据预测，到２０１０年由转基因动物生产的蛋白药物占全部基因工程药物的比率将增长到９５％以上。     

以精子载体法制备转基因小鼠的初步研究

将外源融合基因BaLA-HI与DOSPER脂质体等比较混合,加入获能精子悬液中,37度,5％CO2共培养0．5h,以这种处理精子作为转基因载体乾鼠的体外受精及胚胎移植,在获得的40只移植后代后,经PCR特异片段扩增和Southern杂交,共检测出2只呈相性的转基因小鼠,证明人胰 基因已在小鼠染色体上实现了整合,基因整合率为5％。     

转基因小鼠乳腺表达人乳过氧化物酶的初步研究

从人基因组PAC文库中筛选出人乳过氧化物酶（hLPO）基因,利用长片段PCR的方法获得hLPO基因5’端约3kb片段,通过酶切方法获得hLPO基因3’端约27kb片段,将这两部分拼接并克隆到乳腺特异性表达载体pBC1上,构建以山羊β-casein启动区指导的hLPO的转基因表达载体pBC1-hLPO。利用显微注射的方法获得28只FO代小鼠,经PCR检测和Southerm杂交分析证实,有5只小鼠（4♂,1♀）为整合hLPO基因的转基因阳性小鼠,整合率为17.86％,整合拷贝数在1至5之间。利用SDS-PAGE凝胶电泳和Western blot印迹分析FO、F1代共3只雌性转基因小鼠乳样,结果表明hLPO重组蛋白的特异条带不明显。     

转基因动物检测方法的比较研究

用兔乳清酸性蛋白基因启动子及其远端上游区、免乳清酸性蛋白基因终止子及人瘦蛋白基因cDNA经一系列的亚克隆(Subcloning)构建了用于在转基因动物乳腺中表达人瘦蛋白的特异性表达载体，并采用显微注射的方法制作转基因小鼠动物模型。本文报道在检测转基因小鼠时出现的问题及其解决方案。     

Alteration of the quality of milk by expression of sheep beta-lactoglobulin in transgenic mice

Milk contains a large amount of protein, most of which consists of a few major species synthesized in the mammary gland. The genes encoding these proteins are single-copy, and expressed during pregnancy and lactation. Although beta-lactoglobulin (BLG) is the major protein in the whey of ruminants, it is not present in rodent milk. We have generated transgenic mice carrying the sheep BLG gene, and show that in such mice, BLG is specifically and abundantly expressed in the mammary gland during lactation. This results in a remarkable alteration of milk composition. These findings suggest that the manipulation of milk composition by gene transfer has considerable potential for the improvement of dairy animals.     

EGFP和ALR融合基因表达载体构建及其在HeLa细胞中的表达

从人血液中提取总DNA,利用PCR技术扩增人肝细胞再生增生因子基因,将其插入表达载体pEGFP—C1的多克隆位点中,构建增强绿色荧光蛋白（enhanced green fluorescence protein gene,EGFP）和人肝细胞再生增强因子（human augmenter of liver regeneration gene,ALR）融合基因表达载体pEGFP／ALR,并将其转染Hela细胞系,用含G418的DMEM／F12培养液筛选转基因细胞,然后利用PCR和聚丙烯酰胺凝胶电泳检测转基因细胞中ALR基因的存在及其表达,用荧光显微镜检测EGFP基因的表达．结果显示：得到了构建正确的EGFP和ALR融合基因表达载体;在转基因细胞中,PCR扩增得到1．7Kb的ALR条带,蛋白电泳得到57KD大小条带．与ALR和EGFP融合蛋白大小相符;荧光显微镜下观察到发绿色荧光的Hela细胞．在转基因细胞中,EGFP和ALR同时存在并表达,绿色荧光蛋白可作为报告基因指示目的基因的表达,从而简化了目的基因繁琐的检测手段．     

iRhom2Uncv 小鼠乳腺发育障碍的转录组分析

摘要:目的 研究 iRhom2Uncv 小鼠乳腺表型相关的差异表达基因,为深入研究 iRhom2Uncv 小鼠乳腺发育障碍的相关机制提供靶点信息。 方法 对 12 周龄的野生型和 iRhom2Uncv 小鼠的乳腺组织进行 whole-mounts 染色,分析乳腺发育形态,收集乳腺组织进行转录组测序,筛选差异表达基因,ELISA 检测血清激素水平。 结果 与野生型小鼠相比,iRhom2Uncv 小鼠乳腺侧枝导管较稀疏,差异最显著的 18 个基因中有 6 个在 iRhom2Uncv 小鼠中表达上调,12 个表达下调。 其中 3 个催乳素家族基因在 iRhom2Uncv 小鼠中不表达,且在血清中几乎检测不到催乳素表达,而血清中的雌激素水平相比野生型小鼠较高。 结论 iRhom2Uncv 突变的小鼠存在乳腺发育障碍,可能与 iRhom2Uncv 小鼠中催乳素和 Zfp281 的缺失表达有关,生物信息学分析发现,iRhom2Uncv 小鼠神经发育可能存在不同于野生型小鼠的表型。     

Effect of HS2 and HS3 elements on erythroid-specific expression in transgenic mice

The expression plasmids CMV/GFP, HS2ALL, HS3ALL and HS23ALL were selected to investigate the effect of HS2 and HS3 element on erythroid-specific expression in transgenic mice. These plasmids were digested with restriction enzymes and purified. And five DNA fragments, CMV/GFP, HS2/GFP, CMV/HS2/GFP, HS23/GFP and HS3/GFP were obtained. After purification, the above DNA fragments were microinjected into the pre-nuclei of the mice fertilized eggs and transgenic mice were generated, with an integration rate of 10.89%. The green fluorescence protein(GFP) expression in many transgenic mouse tissues was determined by FACS analysis. The results showed that the HS2 and 1.7 kb of β-globin gene promoter were sufficient for the erythroid-specific expression of β-globin gene. The GFP expression of different recombinant constructs was also analyzed in blood of all the transgenic mice with FACS. The results indicated that HS2 and HS3 had the same enhancement activity on the regulation of β-globin gene expression. Moreover, these two elements showed a significant synergistic effect on gene expression at the transgenic mouse level, although the GFP expression varied largely among different transgenic mouse litters.     

Expression of the intact C<Subscript>4</Subscript> type<Emphasis Type="Italic">pepc</Emphasis> gene cloned from maize in transgenic winter wheat

Maize intact C4-pepc gene was amplified through LA-PCR and successfully sub-cloned into modified vector pGreen0029 to form a stable expression construct named as pBAC214 (12 kb), which contains CaMV 35S promoter driven bar gene as selection marker. Comparing the cloned DNA sequences (6.7 kb) with published maize C4-pepc gene (GenBank accession E17154) sequences, the identity of DNA sequence alignment is 98.96%. There are only 49 differences between these two intact DNA sequences, of which 13 occur in the region of promoter, 18 in introns, and 18 in exons. The homology of mRNA sequence alignment is 99.38%, and the putative amino acids sequence identity is 99.38%. There are only 15 differences between these two mRNA, and these differences bring 4 sites mutant on the putative amino acids of PEPC protein. Through biolistic bombardment of PDS1000/He system, expression vector pBAC214 has been transformed into winter wheat. Southern blotting results show that the intact C4-pepc gene has been integrated into genome of winter wheat. SDS-PAGE analysis of leaf soluble protein in transgenic wheat showed that the intact C4opepc gene was well transcribed, spliced and translated as in maize. The enzyme activity of leaf PEPC in transgenic wheat has been detected. The activities of leaf PEPC increased over 3-5 times in some transgenic plants. The data of photosynthesis rate and transpiration rate of transgenic wheat flag leaves showed that the C4-pepc gene can increase the photosynthesis rate and transpiration rate of transgenic wheat.     

一个在麻疯树根和茎高表达的Kunitz型蛋白酶抑制剂基因JcKTI具有抗虫活性

通过RT-PCR和PCR技术,从麻疯树基因组中克隆得到一个Kunitz型蛋白酶抑制剂基因（JcKTI）的开放阅读框序列。对应开放读码框的基因组序列不含有内含子。该开放阅读框长度为540bp,编码一个含有179个氨基酸残基的成熟多肽,具有典型的Kunitz家族结构特征。组织特异性表达研究显示,JcKTI基因在根和茎中的表达丰度最高,在叶片和种子中表达较低。构建原核表达载体pET32-JcKTI在大肠杆菌BL21中表达,获得纯化的重组蛋白,该蛋白显示出一定的抑制牛胰蛋白酶的活性。将该基因在烟草中过表达,明胶酶法和BAEE法的结果均显示转基因植物的蛋白提取物对胰蛋白酶具有一定抑制作用,进一步的抗虫实验结果表明转基因烟草叶片可使进食后的棉铃虫幼虫生长发育受阻,并减少对叶片的吞食。上述结果暗示JcKTI基因可能在麻疯树根和茎的抗虫应答中扮演着一定的角色。