人溶菌酶基因密码子优化及其在小鼠乳腺中的表达

人溶菌酶是人体内的一种非特异性免疫物质,具有杀菌消炎、免疫调节、改善胃肠道菌群等作用,在抗生素替代方面具有广阔的应用前景。已报道人溶菌酶基因c DNA在内源的αS2酪蛋白基因启动子调控下的表达量不高(23~31μg/m L),特尝试密码子优化策略以改善表达量。优化了人溶菌酶基因c DNA的密码子,连入以小鼠乳清酸蛋白(m WAP)基因座序列为调控元件的乳腺表达载体,验证其表达效率。通过原核注射制备转基因小鼠,PCR检测发现,出生的21只小鼠中有4只为转基因阳性,且能按孟德尔遗传规律传递给下一代;Western blot检测表明,小鼠乳汁中有微量的重组人溶菌酶表达。因此,密码子优化后的人溶菌酶基因可以在小鼠乳腺中获得有效表达,这可为外源基因定点整合的人溶菌酶转基因动物的高效表达研究奠定基础。     

用慢病毒载体介导产生绿色荧光蛋白（GFP）转基因小鼠

以慢病毒（lentivirus）载体为骨架，携带绿色荧光蛋白（GFP）基因的假病毒，通过小鼠受精卵卵周隙注射，将其感染小鼠受精卵，经移植于假孕母鼠后获得转基因小鼠．应用PCR、荧光显微镜观察和流式细胞仪分析等技术，证明了GFP基因的整合率达到40%以上；实时定量PCR分析结果表明转基因小鼠中GFP基因整合的拷贝数约为40；染色体荧光原位杂交分析结果显示GFP基因在小鼠染色体上的整合是随机的，并通过交配可将外源基因遗传至子代，获得的多整合位点和不同表达水平的转基因小鼠具有明显的实际应用和研究价值．文中报道的慢病毒载体介导的转基因技术为高效制备和选育高表达转基因小鼠品系提供了一种有效的途径．     

MicroRNA-122过表达转基因小鼠的建立

目的:建立microRNA-122 (mir-122)过表达转基因小鼠模型,为今后研究肝脏发育,分化及肝癌预防、诊断及治疗等提供实验模型.方法:构建mir-122过表达载体,利用受精卵前核显微注射法将mir-122过表达载体导入小鼠受精卵中,获得mir-122过表达转基因小鼠.PCR法检测转基因小鼠的整合情况,实时定量PCR法检测mir-122过表达转基因小鼠肝脏及其主要器官mir-122及其确定靶基因高亲和性阳离子氨基酸转运体-1(CAT-1),切割同源物1(CUTL-1),血清反应因子(SRF)的表达情况.结果:mir-122过表达转基因小鼠,与同窝阴性鼠相比,mir-122在过表达转基因小鼠肝脏及主要器官中的表达升高,mir-122靶基因表达水平下降.结论:成功构建mir-122过表达转基因小鼠.     

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     

基于Cre／Loxp系统Tyr03、Axl受体酪氨酸激酶转基因小鼠的建立

采用大肠杆菌β一半乳糖酶基因LacZ作为报告基因,建立Tyr03、Axl受体酪氨酸激酶含有loxp位点的转基因小鼠。与不同组织特异性Cre小鼠杂交,获得时空性表达Tyr03、Axl的转基因小鼠,为研究其在各个组织中的功能奠定基础。方法构建含有loxp位点的Tyr03、Axl受体酪氨酸激酶质粒,测序正确后,通过显微注射法将插入CAG启动子下游的Geo—STOP—msAxl及Geo—STOP—msTyr03基因的转基因载体注射入BDFI供体鼠受精卵,移植受精卵至ICR受体鼠,待产仔后,通过双引物PCR鉴定F。以及Fn代小鼠的基因型,RT—PCR,X—gal染色观察小鼠组织中LaeZ基因,运用WB（WesternBlot）检测转基因小鼠和野生型小鼠体内目的蛋白表达的差异。结果获得了含有目的基因的转基因阳性小鼠;通过RT—PCR对阳性小鼠证明LacZ基因的存在;对存在LacZ基因的小鼠进行X—gal染色,在Geo—STOP—msTyr03的脑、睾丸、胸腺中观察到蓝色沉淀,在Geo—STOP—msAxl的脑、心、肾中观察到蓝色沉淀,间接说明目的基因能表达的组织和器官;对双阳性的Geo．STOP．msTyr03、Geo．STOP—msAxl转基因小鼠和野生型小鼠,WB检验证实Tyr03、Axl基因的表达没有差异,说明在加入stop序列之后,Tyr03基因确实存在,而且表达受到抑制。结论成功建立Geo-STOP—msAxl及Geo—STOP—msTyr03酪氨酸受体转基因小鼠。     

肥胖机理初探

机体内有肥胖基因，肥胖基因的表达产物为瘦蛋白，瘦蛋白通过中枢神经系统和内分泌系统调节人的体重，肥胖者是因为“肥胖”的信息不能传至大脑而造成的。     

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．     

小鼠转基因及传代研究

综述了湖北省农科院生物技术研究所近年来有关转基因小鼠的主要研究进展。应用原核注射技术 ,将POMT PGH、hDAF、pBHSA、pSHSA、PT HBV 1 3、Bcl xL、hCD5 9、hCD5 9+hMCP等 8种外源基因 ,注入并移植 4 874枚小鼠的受精卵 ,得到 5 5 6只小鼠 ;经PCR和Southern杂交检测 ,确认原代转基因小鼠 10 8只。基因整合率平均 19.4 % ,转基因效率平均 2 .2 %。应用混合注射的方法得到了转双基因小鼠 ,双基因共整合率 2 2 .2 %。表达外源蛋白的转基因小鼠在 5 0 %～ 10 0 %之间。研究了小鼠的超数排卵和影响转基因效率的几个因素。通过转Bcl xL小鼠与非转基因小鼠连续五个世代的传代交配和检测 ,研究了转基因小鼠外源基因的遗传规律 ,表明四只原代小鼠中 ,只有一只能稳定地将外源基因传递给后代。并非所有的转基因小鼠都具有遗传的稳定性 ,欲建立小鼠的转基因品系 ,尚需对原代转基因小鼠进行筛选。     

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。     

脑组织特异性表达胰岛素样生长因子-1转基因小鼠制备

目的制备脑组织特异性表达胰岛素样生长因子-1(Insulin-Like Growth Factors 1,IGF-1)转基因小鼠。方法采用精子为载体法进行基因转导,出生后小鼠PCR检测,建立转基因小鼠系,用Western blot和免疫荧光法分别检测转基因小鼠海马齿状回亚粒状区(subgranular zone,SGZ)IGF-1表达量和报告基因EGFP阳性细胞数。结果获得3只阳性小鼠,2只外源基因能稳定遗传,并建立了转基因小鼠系,转基因鼠SGZ区域IGF-1表达量显著高于正常鼠,EGFP也在此区域表达。结论成功制备IGF-1转基因小鼠。     

Expression of human erythropoietin directed by mWAP promoter in mammary gland of transgenic mice

The present work has generated transgenic mice with a hybrid gene construct consisting of genomic sequences encodinghuman erythropoietin (hEPO) and governed by regulatory sequences of mousewhey acidic protein (mWAP). The construct proved effective by transient expression in lactating animal. After introducing hybrid gene construct into single-cell embryo via pronuclear microinjection, surviving embryo are reimplanted into pseudopregnant foster mother mouse. 58 mice of 86 generation zero mice obtained were identified to be positive by PCR-Southern blot and genomic DNA Southern blot methods. The integration rate is 67%.hEPO was expressed in the milk of 16 mice of 39 mice measured byhEPO ELISA kit The expression level gets over 15 μg/mL.     

Correction of RNA aberrant splice increases foreign gene expression in transgenic mice

Transgenic mice with mammary gland secreting human granulocyte colony stimulating factor (G-CSF) were produced using mice whey acid protein gene promoter. It was found that there was very low expression level in mammary gland. Human G-CSF cDNA was obtained by RT-PCR from transgenic mice mammary gland. Sequence analysis showed that this G-CSF gene deleted the 4th exon, and compared with human G-CSF genomic DNA, there were donor and acceptor splice sites in the deletion fragment. It was considered that the 3rd and 4th introns also delete in G-CSF fragment. The transgenic construct was corrected by deleting the 3rd and 4th introns to construct the minigene, which was used to produce transgenic mice by microinjection. Northern blot showed that G-CSF expression using the new construct increased 5.4 times as that before in transgenic mice. The results suggested that it was possible that RNA aberrant splice result in low expression in transgenic mice.     

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.     

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.     

有效微注射和移植受精卵建立转基因鼠

试验研究了转基因小鼠建立中的受精卵显微注射和移植，对获得受精卵、有效注射及移植易出现的问题和解决方法进行了讨论，对注射后的受精卵的移植加以改进，建立了人Ｇ－ＣＳＦ转基因小鼠模型．     

牛乳清蛋白BALB/c小鼠过敏模型的建立及其优化

目的:建立牛乳清蛋白的BALB/c小鼠过敏模型,并探讨不同佐剂、免疫途径及饲料纯度等因素对建立牛乳清蛋白的BALB/c小鼠过敏模型的影响。方法:用不含牛乳及乳制品的特制饲料饲养BALB/c小鼠以乳清蛋白致敏,间接ELISA法测定特异性IgE,用荧光法检测体外激发小鼠致敏肥大细胞释放的组胺。结果:成功建立BALB/c小鼠乳清蛋白过敏模型,条件优化结果显示,氢氧化铝佐剂结合皮下注射最好地促进特异性IgE产生;用不含牛奶及其他食物过敏原的特制饲料饲养的小鼠特异性IgE的本底水平低,适于建立小鼠过敏模型,荧光法检测肥大细胞释放组胺的结果与特异性IgE检测结果一致。结论:用特制饲料饲养小鼠,氢氧化铝佐剂结合皮下注射乳清蛋白适于建立BALB/c小鼠过敏症模型,该模型可用于过敏症机理研究。     

Spexin转基因小鼠的构建及表型初步分析

Spexin是一种具有多种生物学功能的神经肽,在代谢疾病及神经系统疾病中发挥重要作用.为了深入研究spexin的生理功能和相关机制,构建了spexin转基因小鼠,通过检测血液中spexin含量以及糖耐受实验,对模型小鼠进行了初步的表型分析.利用piggyBac(PB)转座子技术培育spexin转基因小鼠,采用聚合酶链反应(polymerase chain reaction,PCR)和酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)分别在基因层面和蛋白层面验证了模型动物是否构建成功.之后用饲喂高脂饲料的雄性小鼠进行糖耐受实验,对转基因模型小鼠进行表型分析.PCR结果显示,以靶向spexin的两对引物扩增,转基因型小鼠基因组DNA中能分别扩增出300 bp和301 bp的片段,而野生型小鼠则不能.ELISA结果中,转基因型小鼠血清中spexin含量较野生型小鼠显著升高;同一表型中,雌雄小鼠间血清中spexin水平无明显差异.饲喂高脂饲料85 d内,转基因型小鼠和野生型小鼠体重变化未观察到显著差异;糖耐受结果显示,转基因型小鼠每个时间点血糖浓度均低于野生型小鼠,且在15 min时具有显著性差异.结果显示成功构建了过表达spexin基因的小鼠模型.spexin表达水平的升高在一定程度上提高了C57BL/6小鼠的糖耐受能力.该模型为进一步研究spexin基因在动物体内的功能以及在代谢疾病发病机制中的作用奠定了基础.     

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.