角质细胞特异性表达Cre重组酶转基因小鼠的建立

构建了含有角质细胞特异性角质素5启动子、Cre重组酶基因和人生长激素基因plyA的转基因载体pK5-Cre-hGH。以显微注射的方法将4．2kb的转基因片段K5-Cre-hGH引入小鼠基因组,共注射720枚受精卵,其中龄5枚移植至29只假孕母鼠的输卵管中发育,获得子代小鼠48只,经基因型鉴定有12只小鼠在其基因组上整合有Cre基因,整合率为25％。将带有cre重组酶基因的小鼠与基因组上携带loxP位点的smad4条件基因打靶小鼠杂交以检测Cre重组酶组织特异性表达情况以及介导重组的功能。结果表明,K5-Cre转基因小鼠只在皮肤组织中表达Cre重组酶并能在体内成功地介导loxP位点的重组。     

成骨细胞特异性表达Cre重组酶转基因小鼠的建立

构建了含有骨钙素基因启动子、Cre重组酶基因和人生长激素基因polyA的转基因载体pOC-Cre, 以显微注射的方法将4.6 kb的转基因片段OC-Cre导入小鼠受精卵。16只子代小鼠中经PCR和Southern杂交鉴定, 有2只小鼠携带外源基因, 整合率为12.5%。为了检测OC-Cre在转基因小鼠中表达的组织特异性, 将转基因首建者小鼠与基因组上携带有LoxP位点的条件性Smad4基因敲除小鼠交配, PCR结果显示, 仅在子代纯合型小鼠骨组织基因组中扩增出了Cre介导重组后的片段。将OC-Cre转基因小鼠与ROSA26报告小鼠交配, 利用LacZ染色对双转基因阳性子代小鼠进行检测, 结果显示Cre重组酶在成骨细胞中特异性表达并介导ROSA基因座LoxP位点间的重组。所有这些结果说明：所建立的OC-Cre转基因小鼠在成骨细胞中特异性表达Cre重组酶, 并能在体内介导成骨细胞基因组上LoxP位点间的重组, 是一种理想的研制成骨细胞特异性基因敲除小鼠的工具小鼠。     

Ⅱ型肺泡细胞特异表达SARS冠状病毒核衣壳蛋白转基因小鼠的建立

目的:建立Ⅱ型肺泡细胞特异表达SARS冠状病毒(SARS-CoV)核衣壳蛋白(N蛋白)的转基因小鼠。方法:用分子克隆的方法构建包括肺表面活性蛋白A(SP-A)启动子、SARS-CoVN蛋白基因、β-半乳糖苷酶(LacZ)报告基因和人生长激素(hGH)polyA的转基因载体pSP-A-N。以显微注射的方法将8.3kb的转基因片段引入小鼠受精卵。通过PCR、Southern印迹和LacZ染色检测子代小鼠中转基因的整合及表达。结果:共注射952枚受精卵,移植至42只假孕母小鼠的输卵管中发育,获得子代小鼠128只,经PCR、Southern印迹鉴定,其中11只小鼠基因组上整合有SARS-CoVN蛋白基因,整合率为8.6%。鉴定结果显示,11只转基因首建者小鼠中有1只表达外源基因并能正常传代。LacZ染色结果表明N蛋白基因在转基因小鼠Ⅱ型肺上皮细胞中特异表达。结论:成功构建了Ⅱ型肺泡细胞特异表达SARS-CoVN蛋白的转基因小鼠,为深入研究该基因的病理生理学效应奠定了基础。     

外源基因在转基因小鼠中遗传和表达的稳定性

通过显微注射将外源构件λ１０６（年α－Ｓ１酪蛋白基因调控区指导的乙肝病毒表面抗原基因表达构件）导入小鼠受精卵中,用ＰＣＲ－Ｓｏｕｔｈｅｍ方法进行外源基因的整合检测、ＥＬＩＳＡ法进行外源基因的表达检测,对４代转基因小鼠进行了跟踪测定,结果表明：的代转基因小鼠的整合率为５６％,原代雌鼠乳汁中转基因产物的表达率为１００％;传代跟踪基因的整合率不符合“单一位点随机整合”的假说,家系分析推测外源基因在受精卵     

鲤鱼生长激素基因在鲫鱼中的基因转移及PCR检测

鲫鱼群体内,个体之间的生长速度差别不大,遗传保守性较强,用常规育种方法很难获得生长速度快、个体差异较大的后代。通过基因转移技术,将外源基因如鱼的生长激素基因导入鱼的受精卵中,而获得转基因鱼是钱定向育种的一条有效途径。已有报道,在虹鳟鱼生长激素cDNA上游连结一个病毒启动子构建成表达质粒,由此获得的转基因鲤鱼比对照生长速度快20％。但是未见有将鲤鱼生长激素基因导入鲫鱼受精卵的报导。我们克隆了鲤鱼生长激素基因,并用它构建了含有病毒启动子（F.1,插入0．8Kb,位于XbaI和PstI之间)的表达质粒（Fig.2,命名为pCMV－TK－CGH）。用该质粒对鲫鱼受精卵进行注射,注射了1000粒卵,获得了转基因实验鲫鱼480尾。孵化率为48％。将其中100尾幼鱼于水簇箱中进行放养。选择其中个体较大的50尾进行PCR检测。其中8条鱼的基因组DNA有0．6Kb的PCR扩增产物（Fig.3),表明其上整合有外源的鲤鱼生长激素基因,其整合率为16％。其中最大的一条体重4．2g,体和6cm,比对照（0．7g,4cm）体重增加5倍,体长增加2cm(Fig.4)。我们构建的是高效表达质粒,其上含有巨细胞病毒增强子和人单纯疱疹病毒胸苷激酶启动子（属强启动子）,对鲤鱼生长激素基因有较强的启动作用。鲤鱼和鲫鱼在分类上属同科不同属,考虑到两者生长激素基因有较强的启动作用。鲤鱼和鲫鱼在分类上属同科不同属,考虑到两者生长激素基因的同源性,对转基因鲫鱼的PCR检测参照了加拿大学者Du所用引物的设计、使注射的外源基因与其本身的内源生长激素基因相区别。我们的转基因整合率（16％）与Zhang(10%)、Grass和Hackett等报道的接近。转基因鲫鱼生长速度增加的倍数（5倍）与Du等人的结果（2－6）接近。但是,这一生长速度快的优势能否遗传给后代,有待进一步观察。     

鲤鱼和草鱼基因文库的构建及其生长激素基因和肌动蛋白基因的筛选

用显微注射方法,把由小鼠重金属鳌合蛋白(MT)基因启动子驱动的人生长激素(hGH)基因导人鲤鱼等的受精卵内,由此发育而成的一部分鱼的基因组内携带有MThGH基因,这些鱼称之为"转基因鱼"1.       

重组人apoE基因表达活性和转基因小鼠研究

人apoE基因组DNA,去除其自身启动子,代之以小鼠金属硫蛋白启动子,重组质粒经脂质体介导转入小鼠NIH／3T3细胞后,以人apoE基因组DNA／EcoRⅠ片段为探针检测mRNA表达,可见apoEmRNA杂交信号很强,经重金属诱导后杂交信号更强,表明MT启动子功能良好,pME表达正常．将人apoE基因组DNA用显微注射法导入小鼠受精卵雄性原核,再将胚胎移植入假孕母鼠输卵管内,仔鼠分娩四周后,自鼠尾提取DNA,鉴定人apoEDNA在小鼠染色体上的整合,最终得到有人apoE基因整合的转基因首建鼠．     

鲤鱼生长激素基因在鲫鱼中的基因转移及PCR检测

鲫鱼群体内,个体之间的生长速度差别不大,遗传保守性较强,用常规育种方法很难获得生长速度快、个体差异较大的后代。通过基因转移技术,将外源基因如鱼的生长激素基因导入鱼的受精卵中,而获得转基因鱼是鱼类定向育种的一条有效途径。已有报道,在虹蹲鱼生长激素ｃＤＮＡ上游连结一个病毒启动子构建成表达质粒,由此获得的转基因鲤鱼比对照生长速度快２０％。但是未见有将鲤鱼生长激素基因导入鲫鱼受精卵的报导。我们克隆了鲤鱼生长激素基因,并用它构建了含有病毒启动子（Ｆｉｇ．１,插入片段为０．８Ｋｂ,位于ＸｂａＩ和ＰｓｔＩ之间）的表达质粒（Ｆｉｇ．２,命名为ｐＣＭＶ－ＴＫ－ＣＧＨ）。用该质粒对鲫鱼受精卵进行注射,共注射了１０００粒卵,获得了转基因实验鲫鱼４８０尾,孵化率为４８％。将其中１００尾幼鱼于水簇箱中进行放养。选择其中个体较大的５０尾进行ＰＣＲ（检测。其中８条鱼的基因组ＤＮＡ有０．６Ｋｂ的ＰＣＲ扩增产物（Ｆｉｇ．３）,表明其上整合有外源的鲤鱼生长激素基因,其整合率为１６％。其中最大的一条体重４．２ｇ,体长６ｃｍ,比对照（０．７ｇ,４ｃｍ）体重增加５倍,体长增加２ｃｍ（Ｆｉｇ．４）。我们构建的是高效表达质粒,其上含有巨细胞病毒增强子和     

胰腺组织表达Cre重组酶转基因小鼠的建立及鉴定

组织特异性表达Cre重组酶的转基因小鼠是进行组织特异性基因剔除研究的重要工具。为了建立胰腺组织特异性Cre转基因小鼠,我们通过PCR克隆了大鼠胰岛素基因启动子,并用它指导Cre基因在胰岛细胞中的特异性表达。在Cre重组酶基因5′端添加了真核核糖体结合序列和核定位序列以使Cre重组酶能穿越核膜在细胞核中发挥功能;同时,在Cre基因3′端添加了含内含子的3′端人生长激素基因。表达载体经显微注射导入小鼠受精卵以建立转基因小鼠。PCR检测显示共获得7只Cre整合阳性的转基因首建者小鼠;RTPCR结果表明其中1只首建者小鼠的子代鼠在胰腺中转录了外源基因,进一步的Southern杂交结果表明,该转基因小鼠能够在胰腺中表达有功能的Cre重组酶。      

中枢神经系统特异性表达Cre重组酶的转基因小鼠

利用从129sv小鼠基因组文库克隆得到的1．8kb的胶质细胞原纤维酸性蛋白(GFAP)基因的5′端调控序列,构建了含有2个β—珠蛋白绝缘子、GFAP5′端调控区、Cre基因和人生长激素基因(hGH)polyA的转基因载体pGFAP—Cre—hGH。以显微注射的方法将7．6kb的转基因片段pGFAP—Cre—hGH引入191枚小鼠基因组受精卵,其中176枚分别移植至8只假孕母鼠的输卵管中使其发育,共获得子代小鼠25只。经PCR和Southern杂交鉴定其中7只小鼠基因组上整合有Cre基因,整合率为28％。用整合有Cre基因的转基因小鼠与基因组上整合有LoxP位点和LacZ表达框的ROSA26鼠杂交,以检测Cre酶的活性、组织特异性及其介导的两个LoxP位点间的重组。LacZ染色结果表明,GFAP—Cre转基因小鼠只在中枢神经系统中表达Cre重组酶并能在体内成功介导LoxP位点间的重组。     

Analysis of transgenic tobacco plants carrying the gene for the surface antigen of the hepatitis B virus

The plasmids carrying the gene encoding the hepatitis B surface antigen (HBsAg) under the control of 35S RNA single or dual promoters of the cauliflower mosaic virus CaMV 35S were constructed. These constructions were used for obtaining transgenic tobacco plants that synthesize the HBS antigen. The presence of HBsAg in tobacco plant extracts was confirmed by the enzyme-linked immunoassay using antibodies against the native HBs antigen. The antigen amount in plants carrying the HbsAg gene under a single 35 S promoter was 0.0001-0.001 of the total soluble protein whereas the use of a dual 35S promoter increased the antigen synthesis to 0.002-0.05% of the protein. The antigen-synthesizing ability was inherited by the offspring. In the F1 plants, the antigen expression varied in different lines comprising 0.001 to 0.03% of the total soluble protein, which corresponded to the antigen amount in the F0 plants.     

In vitro and in vivo synthesis of the hepatitis B virus surface antigen and of the receptor for polymerized human serum albumin from recombinant human adenoviruses.

We have developed an adenovirus vector to express foreign proteins under the control of the adenovirus E1a promoter. Two recombinant plasmids, harbouring either the S gene or the pre-S2 region and the S gene of hepatitis B virus under the control of the E1a promoter, were used to construct two recombinant adenoviruses. These two viruses direct the synthesis of hepatitis B virus surface antigen (HBsAg) particles during the time course of an infectious cycle. When the pre-S2 region is present in the constructed virus, the synthesis of particles carrying the receptor for polymerized human serum albumin (pHSA) is observed. Moreover, the inoculation of rabbits with this latter purified recombinant adenovirus elicits the production of antibodies that react with both HBsAg and pHSA receptor.     

Analysis of Transgenic Tobacco Plants Carrying the Gene for the Surface Antigen of the Hepatitis B Virus

The plasmids carrying the gene encoding the hepatitis B surface antigen (HBsAg) under the control of 35S RNA single or dual promoters of the cauliflower mosaic virus CaMV 35S were constructed. These constructions were used for obtaining transgenic tobacco plants that synthesize the HBs antigen. The presence of HBsAg in tobacco plant extracts was confirmed by the enzyme-linked immunoassay using antibodies against the native HBs antigen. The antigen amount in plants carrying the HBsAg gene under a single 35S promoter was 0.0001–0.001 of the total soluble protein whereas the use of a dual 35S promoter increased the antigen synthesis to 0.002–0.05% of the protein. The antigen-synthesizing ability was inherited by the offspring. In the F₁ plants, the antigen expression varied in different lines comprising 0.001 to 0.03% of the total soluble protein, which corresponded to the antigen amount in the F₀ plants.     

Enhanced ascorbic acid accumulation in transgenic potato confers tolerance to various abiotic stresses

l-Ascorbic acid (Vitamin C, AsA) is an important component of human nutrition. Plants and several animals can synthesize their own ascorbic acid, whereas humans lack the gene essential for ascorbic acid biosynthesis and must acquire from their diet. In the present study, we developed transgenic potato (Solanum tuberosum L. cv. Taedong Valley) over-expressing l-gulono-γ-lactone oxidase (GLOase gene; NCBI Acc. No. NM022220), isolated from rat cells driven by CaMV35S constitutive promoter that showed enhanced AsA accumulation. Molecular analyses of four independent transgenic lines performed by PCR, Southern and RT-PCR revealed the stable integration of the transgene in the progeny. The transformation frequency was ca. 7.5% and the time required for the generation of transgenic plants was 6–7 weeks. Transgenic tubers showed significantly enhanced AsA content (141%) and GLOase activity as compared to untransformed tubers. These transgenics were also found to withstand various abiotic stresses caused by Methyl Viologen (MV), NaCl or mannitol, respectively. The T₁ transgenic plants exposed to salt stress (100 mM NaCl) survived better with increased shoot and root length when compared to untransformed plants. The elevated level of AsA accumulation in transgenics was directly correlated with their ability to withstand abiotic stresses. These results further demonstrated that the overexpression of GLOase gene enhanced basal levels of AsA in potato tubers and also the transgenics showed better survival under various abiotic stresses.     

Enhanced viral resistance in transgenic mice expressing the human beta 1 interferon.

Recombinant plasmids pMTIF-beta 1A and pMTIF-beta 1B were constructed by fusing the metallothionein I promoter-regulatory region to the human beta 1 interferon (HuIFN-beta 1) gene. These linearized fusion genes were then introduced into mouse germ lines by zygote microinjection. The chromosomal integration and the germ line transmission of the injected DNA sequences in the resulting transgenic mice were detected by DNA dot blot and Southern transfer hybridizations. The sera of at least two strains of metallothionein/HuIFN transgenic mice were found to protect human WISH cells against vesicular stomatitis virus infection, and this activity could be neutralized by preincubation with anti-HuIFN-beta 1 antibody. These transgenic mice demonstrated significantly enhanced resistance to pseudorabies virus compared with nontransgenic mice when inoculated with pseudorabies virus. The level of resistance seemed to correlate with the concentrations of HuIFN-beta 1 in serum. These transgenic mice may be used as models to study IFN-induced responses and may serve as prototypes to generate disease-resistant animals.     

Construction and characterization of hybrid hepatitis B antigen particles carrying a poliovirus immunogen

The hepatitis B surface antigen (HBsAg) has the unique property of assembling with cellular lipids into spherical or elongated particles of 22 nm diameter which are secreted by mammalian cells expressing HBsAg. We have studied the structural requirements for particle formation and secretion by creating in-phase insertions into different regions of the S gene of the hepatitis B virus, coding for HBsAg. Modified genes were integrated into an appropriate vector and expressed in mouse L cells. Various single and double inserts in the two major hydrophilic domains of HBsAg were compatible with particle synthesis and secretion. The level of secretion was influenced by the length of the insert, its primary structure, and the site of insertion into the HBsAg molecule. One of the inserted sequences was a synthetic DNA fragment encoding a continuous type 1 poliovirus neutralization epitope (the C3 epitope). Mammalian cells expressing the modified hepatitis B virus S gene secreted hybrid particles carrying the poliovirus antigen. The hybrid polio-HBsAg particles reacted with a monoclonal antibody specific for the C3 epitope and induced poliovirus neutralizing antibodies at low, but significant, titers in mice and at high titers in rabbits. However, the immune response to HBsAg was weaker to hybrid particles than to unmodified HBsAg particles. By cotransfection with two different plasmids carrying either modified or unmodified genes, we obtained phenotypically mixed particles containing both polio-HBsAg and HBsAg molecules. Inoculated into rabbits, the mixed particles induced high antibody titers against both poliovirus and HBsAg.     

Life history of the bird cherry-oat aphid, Rhopalosiphum padi (Homoptera: Aphididae), on transgenic and untransformed wheat challenged with barley yellow dwarf virus

The life history of Rhopalosiphum padi (L.) was monitored on transgenic and untransformed (soft white winter wheat plants that were infected with Barley yellow dwarf virus (BLDV), noninfected, or challenged with virus-free aphids under laboratory conditions. Two transgenic soft white winter wheat genotypes (103.1J and 126.02) derived from the parental variety Lambert and expressing the barley yellow dwarf virus coat protein gene, and two untransformed varieties, virus-susceptible Lambert and virus-tolerant Caldwell, were tested. B. padi nymphal development was significantly longer on the transgenic genotypes infected with BYDV, compared with noninfected transgenic plants. In contrast, nymphal development on Lambert was significantly shorter on BYDV-infected than on noninfected plants. Nymphal development on noninfected Lambert was significantly longer than on noninfected transgenics. No significant difference in nymphal development period was detected between virus-infected and noninfected Caldwell. Aphid total fecundity, length of reproductive period, and intrinsic rate of increase were significantly reduced on BYDV-infected transgenic plants compared with BYDV-infected Lambert. In contrast, reproductive period, total adult fecundity, and intrinsic rate of increase on noninfected Lambert were significantly reduced compared with noninfected transgenics. Transgenic plants infected with BYDV were inferior hosts for R. padi compared with infected Lambert. However, noninfected transgenics were superior hosts for aphids than noninfected Lambert. Moderate resistance to BYDV, as indicated by a significantly lower virus titer, was detected in the transgenic genotypes compared with the untransformed ones. Results show for the first time that transgenic virus resistance in wheat can indirectly influence R. padi life history.     

J亚群禽白血病病毒NX0101株的TCID50与p27抗原之间的相关性研究

为了研究J亚群禽白血病病毒在细胞上接种后的半数细胞培养物感染量(TCID₅₀)与p27抗原的酶联免疫吸附试验（ELISA）的S/P值之间的相关性,并探讨其意义。本试验将J亚群禽白血病病毒NX0101株接种鸡胚成纤维细胞（CEF细胞）,换维持液后连续10d取样,检测10d的TCID₅₀值与p27抗原的S/P值之间的相关性。同时,将该毒株在DF-1细胞系上传代至20代,取其中的第1代、第5代、第10代、第15代和第20代分别进行TCID₅₀滴度的测定和p27抗原检测。结果表明:在CEF细胞上接种的NX0101株J亚群禽白血病病毒连续10d的TCID₅₀值与p27抗原之间存在显著的相关性（r=0.85277;P<0.0001）呈正相关;在DF-1细胞系上传的不同代数之间也呈显著正相关（r=0.93000;P=0.0220）。由此可以推测J亚群禽白血病病毒的TCID₅₀与p27抗原呈显著正相关,因而可以用ELISA法测得的p27抗原的S/P值对病毒的TCID₅₀值进行估测。     

Production of marker-free plants expressing the gene of the hepatitis B virus surface antigen

The pBM plasmid, carrying the gene of hepatitis B virus surface antigen (HBsAg) and free of any selection markers of antibiotic or herbicide resistance, was constructed for genetic transformation of plants. A method for screening transformed plant seedlings on nonselective media was developed. Enzyme immunoassay was used for selecting transgenic plants with HBsAg gene among the produced regenerants; this method provides for a high sensitivity detection of HBsAg in plant extracts. Tobacco and tomato transgenic lines synthesizing this antigen at a level of 0.01–0.05% of the total soluble protein were obtained. The achieved level of HBsAg synthesis is sufficient for preclinical trials of the produced plants as a new generation safe edible vaccine. The developed method for selecting transformants can be used for producing safe plants free of selection markers.