通用型基因打靶载体的构建及其功能鉴定

为了构建适合大多数基因座位点打靶的通用型基因打靶载体及打靶成功后去除正选择标记基因,以克隆载体pGEM-3Z为骨架,插入了一个正选择标记基因新霉素磷酸转移酶基因(neo).两个相同的负选择标记基因单纯疱疹病毒胸苷激酶基因HSV-tk1和HSV-tk2,并在neo的两侧各添加了一个方向相同的LoxP(10cus of crossing-over(X)in P1)序列及两个不同的多克隆位点序列,从而构建了载体pA2T.插入的两个不同的多克隆位点序列中,neo和HSV-tk1之间的多克隆位点序列有8个稀少的酶切位点、neo和HSV-tk2之间的多克隆位点序列有5个稀少的酶切位点,neo、HSV-tk1和HSV-tk2有各自独立的转录单元.脂质体法转染山羊成纤维细胞,用遗传霉素(G418)和丙氧鸟苷(GAC)进行正负筛选,验证了正负选择标记基因的生物活性,证明通用型基因打靶载体pA2T构建成功.栽体pA2T转化组成性表达Cre重组醇(Cyclization recombination protein)的大肠杆菌BM25.8,检测到LoxP序列的生物活性,结果表明pA2T中的正选基因可以被Cre重组酶去除.因此,本研究所构建的通用型基因打靶载体pA2T,根据不同的基因座设计同源臂后,插入到MCS中可直接用于不同基因座位点的打靶,并能够在打靶成功后用Cre重组酶去除基因组中插入的neo基因,为用基因打靶的方法制作转基因动物提供了便利.     

Expression of the hIGF-I gene driven by the Fhx/P25 promoter in the silk glands of germline silkworm and transformed BmN cells

The expression of the human insulin-like growth factor (hIGF-I) gene driven by the Fhx/P25 promoter in the silk glands of transgenic silkworms (Bombyx mori) and in transformed silkworm cells, was achieved using BmN cells transfected with a piggyBac vector, pigA3GFP-Fhx/P25-hIGF-ie-neo containing a neomycin-resistance gene (neo), a green fluorescent protein gene (gfp), an hIGF-I gene, and a helper plasmid containing the piggyBac transposase sequence under the control of the B. mori actin 3 (A3) promoter. We selected stably transformed BmN cells expressing hIGF-I using the antibiotic G418. The expression level of hIGF-I was about 450 pg in 3 × 10(6) cells, determined by ELISA. The piggyBac vector was transferred into the silkworm eggs using sperm-mediated gene transfer. The expression level of hIGF-I per gram fresh posterior silk glands of G4 transgenic silkworms was approx. 150 ng.     

Acetamide selection of Kluyveromyces lactis cells transformed with an integrative vector leads to high-frequency formation of multicopy strains

The yeast Kluyveromyces lactis has been extensively used as a host for heterologous protein expression. A necessary step in the construction of a stable expression strain is the introduction of an integrative expression vector into K. lactis cells, followed by selection of transformed strains using either medium containing antibiotic (e.g., G418) or nitrogen-free medium containing acetamide. In this study, we show that selection using acetamide yields K. lactis transformant populations nearly completely comprised of strains bearing multiple tandem insertions of the expression vector pKLAC1 at the LAC4 chromosomal locus, whereas an average of 16% of G418-selected transformants are multiply integrated. Additionally, the average copy number within transformant populations doubled when acetamide was used for selection compared to G418. Finally, we demonstrate that the high frequency of multicopy integration associated with using acetamide selection can be exploited to rapidly construct expression strains that simultaneously produce multiple heterologous proteins or multisubunit proteins, such as Fab antibodies.     

利用启动子缺陷型打靶载体敲除五指山小型猪GGTA1基因

目的:获得α-1,3-半乳糖基转移酶(GGTA1)基因敲除的五指山小型猪胎儿,为异种器官移植研究提供基础平台。方法:以新霉素磷酸转移酶基因(neo)为筛选标记基因构建启动子缺陷型打靶载体,打靶载体线性化后用电转染法将其导入胎儿成纤维细胞中,转染后的细胞经G418筛选后,用PCR法检测药物抗性的细胞克隆,对阳性细胞进行核移植构建重构胚胎及胚胎移植。结果:转染后,经筛选共得到176个具有G418抗性的细胞克隆,经PCR检测,其中2个细胞克隆发生了同源重组;以其中1个GGTA1+/-细胞克隆为供体细胞进行核移植,将重构胚移植到2头自然发情的受体母猪中,1头受体妊娠;第37 d将代孕母猪处死,获得2个胎儿,经PCR和Southern印迹鉴定,均为GGTA1单等位基因敲除胎儿。结论:构建了五指山小型猪GGTA1基因部分外显子4区域敲除的启动子缺陷型打靶载体,获得了GGTA1单等位基因敲除的胎儿,为培育GGTA1基因敲除的五指山小型猪奠定了基础。     

Induced recombination between duplicated neo genes stably integrated in the genome of CHO cells

Homologous recombination between 2 truncated neo genes stably integrated in the genome of Chinese hamster ovary (CHO) cells was studied. A vector containing a functional gpt gene and 2 tandemly arranged G418 resistance (neo) gene fragments with about 400 bp of sequence homology was transfected into CHO cells. Clonal cell lines were established from transfected cultures and the spontaneous frequency of G418-resistant revertants was found to range between 1 x 10(-4) and 5 x 10(-4). The ability of the alkylating agents MMS and HN2 to induce recombination of the transfected neo genes was studied in 2 of the cell lines. After treatment with MMS at doses that reduced survival to 10% of the control these cell lines showed a dose-dependent increase in the frequency of G418-resistant revertants. No effect was observed after treatment with HN2. All G418-resistant subclones contained a new restriction fragment indicating that a whole neo gene had been formed by rearrangement in pairs of truncated neo genes. Hence, this system can be used to study molecular mechanisms and chemical inducibility of homologous recombination in mammalian cells.     

以新霉素抗性基因突变体为筛选标志的真核表达载体的构建

新霉素抗性基因(neo)是真核表达载体的常用筛选标志neo基因编码新霉素磷酸转移酶Ⅱ（NPT Ⅱ）,能催化G418、卡那霉素等多种氨基糖苷抗生素分子磷酸化而使之失去抗菌活性。通过对真核表达载体的筛选标志基因neo进行定点突变,以降低NPTⅡ的活性,然后用含neo突变体的真核表达载体pmDNA构建荧光素酶表达质粒,稳定转染CHO-K1细胞,发现表达荧光素酶的阳性细胞比例达到95%,其中高表达细胞集落的筛选率明显高于对照组。     

甲醇酵母表达系统高拷贝数整合型表达载体的构建

以甲醇酵母(Hansenula polymorpha)表达系统的Yip型表达载体pJF5-1为基础,通过引入宿主来源的一个rDNA随机顺序和由SV40早期启动子控制的G418抗性基因(neo)及对野生型标记基因Hpleu2启动子大部分上游序列的缺失,构建了一个高拷贝整合型表达载体pMIRH。有关转化、筛选和拷贝分析等试验结果表明,由pMIRH可产生含高拷贝数载体的转化子,并可通过由leu2+筛选和G418抗性筛选所组成的二级筛选方法富集这些含高拷贝数载体的转化子。有关完整DNA和消化DNA Southern杂交分析结果进一步表明,上述高拷贝数的载体以串联重复排列的方式整合于宿主基因组。     

Introduction of new genetic material into human myeloid leukemic blast stem cells by retroviral infection.

An amphotropic retroviral vector containing the bacterial neomycin phosphotransferase gene (neo) was used to infect blast cells from patients with acute myeloblastic leukemia. The infected cells acquired a G418-resistant phenotype that was stable as measured in a clonogenic assay and in long-term suspension culture. Thus, gene transfer into stem cells was accomplished by this procedure. This approach for manipulating gene expression in blast stem cells provides a means to assess the roles of a variety of genes in self-renewal, differentiation, and leukemogenesis.     

Acetamide Selection of Kluyveromyces lactis Cells Transformed with an Integrative Vector Leads to High-Frequency Formation of Multicopy Strains

The yeast Kluyveromyces lactis has been extensively used as a host for heterologous protein expression. A necessary step in the construction of a stable expression strain is the introduction of an integrative expression vector into K. lactis cells, followed by selection of transformed strains using either medium containing antibiotic (e.g., G418) or nitrogen-free medium containing acetamide. In this study, we show that selection using acetamide yields K. lactis transformant populations nearly completely comprised of strains bearing multiple tandem insertions of the expression vector pKLAC1 at the LAC4 chromosomal locus, whereas an average of 16% of G418-selected transformants are multiply integrated. Additionally, the average copy number within transformant populations doubled when acetamide was used for selection compared to G418. Finally, we demonstrate that the high frequency of multicopy integration associated with using acetamide selection can be exploited to rapidly construct expression strains that simultaneously produce multiple heterologous proteins or multisubunit proteins, such as Fab antibodies.     

Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells.

We describe the construction of an adeno-associated virus (AAV) vector in which the coding sequence of the procaryotic gene neo is expressed under the control of the major AAV promoter p40. This AAV-neo vector allowed stable expression of neo as a dominant selective marker in mammalian cells by selection of cells which were resistant to the antibiotic geneticin (G418). When the vector was introduced into human (293 or HeLa) cell lines by a DNA transfection procedure, stable geneticin-resistant colonies were obtained. When the vector was first packaged into AAV particles and then introduced into cells via particle infection, geneticin-resistant cells were obtained at higher frequencies than those obtained by DNA transfection. In geneticin-resistant cells the AAV-neo vector was integrated at low copy number and could be rescued by subsequent infection with wild-type AAV and the helper adenovirus or, in some cases, by infection with adenovirus alone. The rescued AAV-neo vector could then be recovered as amplified unintegrated DNA from a Hirt lysate. These results demonstrate that AAV can be used as a transducing viral vector for stable integration and expression of a foreign gene in mammalian cells. The high frequency of integration and the ability to rescue the integrated vector suggest that this vector system may be useful for selecting genes from cDNA libraries. This vector may also be useful for introduction of genes into cells which are refractory to transfection in procedures such as those involving the use of CaPO4 or DEAE-dextran.     

G418抗性HEK293细胞的培育

目的　培育具有G418抗性的HEK2 93细胞 ,用于建立猪内源性反转录病毒感染人HEK2 93细胞的模型。方法　通过脂质体转染的方法 ,将含有neo基因的质粒pIRESneo导入HEK2 93细胞中 ,利用G418的选择特性 ,对转染细胞进行压力筛选 ,并对其进行了PCR鉴定。结果　经 6 0 0 μg ml的G418压力筛选后 ,获得了抗性细胞克隆。抗性细胞的形态和生长速度与筛选前细胞没有差异 ,特异性核苷酸引物检测抗性细胞基因组DNA ,可以扩增出对应的核苷酸片段。结论　成功地培育了G418抗性HEK2 93细胞 ,为建立猪内源性反转录病毒感染人HEK2 93细胞的模型奠定了基础。     

狂犬病病毒糖蛋白哺乳动物细胞稳定表达细胞系建立

试验首先根据GenBank上所发表的狂犬病病毒CVS-24株糖蛋白基因序列,设计并合成一对特异性引物,通过反转录 聚合酶链式反应（RT-PCR）,获得糖蛋白全长cDNA,连接在pMD18-T载体上,测序证明克隆的正确性后,将其插入真核表达载体pIRES1neo,构建了糖蛋白单一表达载体pICG,表达质粒通过脂质体转染BHK-21细胞,在G418抗性压力下出现细胞克隆,通过PCR检测,确定启动子与糖蛋白基因在细胞基因组中共同整合。借助Western blot检测,证明所表达的糖蛋白与狂犬病抗血清有特异的反应性。采用间接ELISA法,筛选出3株高效表达糖蛋白的细胞株,分别命名为ICG1、ICG2、ICG3。     

Enhanced growth in NS0 cells expressing aminoglycoside phosphotransferase is associated with changes in metabolism, productivity, and apoptosis

Prior work has reported that cotransfecting a gene of interest with the selectable marker neo can seriously perturb a number of cellular processes. In this study the influence of the neo gene on the growth, death, and metabolism of a murine myeloma NS0 cell line, expressing a chimeric antibody, was investigated. A pool of neo transfectants, 6A1-NEO, was selected with 500 microg/mL G418. Quantitative PCR analysis revealed that 6A1-NEO contained, on average, three copies of the neo gene per cell. Batch cultivation of 6A1-NEO showed that there was a 36% increase in maximum viable cell concentration, a 20% increase in the maximum apparent growth rate, and a 134% increase in cumulative cell hours as compared with the parent, 6A1-(100)3. Batch cultivation of five randomly selected clones illustrated that 6A1-NEO's advantage over the parent was not due to clonal variation. Neither the use of G418 during the selection process nor the cultivation of cells in the presence of G418 were responsible. This implied that the neo gene product, APH(3')-II, was causing the changes in proliferative capacity. Analysis of the cell cycle revealed that there were no differences in the distribution of cells in the G(1), S, and G(2) phases. When cell growth was synchronized, there were no observed differences in cell-cycle duration. 6A1-NEO resisted the onset of apoptosis during the growth phase. Consequently, there was a larger viable population of 6A1-NEO cells available for proliferation as compared with the parent. However, 6A1-NEO died at the same rate as the parent when resuspended in spent media or after treatment with staurosporin. Expression of the anti-apoptotic protein Bcl-2 was upregulated in 6A1-NEO, indicating that APH(3')-II could be acting by modulating endogenous gene expression. Analysis of key metabolites showed that 6A1-NEO's specific glucose consumption rate was 133% higher, whilst its specific glutamate consumption rate was 45% lower than the parent. 6A1-NEO's efficient utilization of glutamate and shift towards glucose metabolism may have contributed to the rise in proliferative capacity. However, this was accompanied by a 70% drop in the specific antibody production rate. These results show that the increase in growth rate and proliferative capacity caused by the expression of recombinant APH(3')-II was associated with changes in metabolism, apoptosis, and endogenous gene expression.     

细胞粘附分子P-selectin功能片段在CHO细胞表面的锚定表达

为了筛选出针对细胞粘附分子P-selectin的特异性抗体, 克隆了P-selectin功能性基因片段, 使其通过糖基化磷脂酰肌醇(GPI)锚定表达于真核细胞的细胞膜上, 以用作筛选的抗原。提取人血小板的总RNA, RT-PCR扩增出P-selectin目的基因片段, 同时用重叠延伸PCR的方法合成细胞膜锚定信号肽GPI基因; 将二者按上下游顺序插入含有弱化neo基因的真核表达载体pMCEw2中; 并将构建的重组质粒pMCEw2-GPI-P-selectin转染CHOdhfr-细胞, G418筛选获得阳性细胞株, 利用ELISA、免疫印迹和免疫荧光法检测P-selectin的表达。实验证实了P-selectin在细胞膜上获得稳定表达。为进一步进行抗P-selectin的特异性抗体的筛选提供了必要前提。     

以neo作选择标记富集和筛选阳性重组杆状病毒

将苜蓿银纹夜蛾(AcNPV)极早期基因IEI启动子控制的neo基因表达盒插入p10型转载体pAcEP106中得到pAcPIneo,将它和野生型AcNPV DNA共转染Sf细胞得到neo^+重组病毒.因为neo基因的表达使得neo^+基因的重组病毒可以在G418的存在下复制,而野生型则不能.将共转染得到的上清液以很低的感染复数连续传代,通过G418的选择作用,使重组病毒得到富集,三代以后重组病毒比例可达90%以上,如此之高的重组比例使得重组病毒的筛选不须经空斑纯化只须有限稀释即可得到.利用杆状病毒早期基因启动子表达neo基因为用早期基因表达外源毒素基因作重组病毒杀虫剂打下了基础,同时也建立了一种简便有效的筛选、富集阳性重组病毒的方法.     

Nature and specificity of lymphokine independence induced by a selectable retroviral vector expressing v-src.

A murine retroviral vector, LSNLsrc, has been constructed and examined for its ability to induce growth factor independence in cells normally dependent on interleukin 2 (IL-2) or interleukin 3 (IL-3) for growth. The LSNLsrc vector coexpressed the v-src gene of Rous sarcoma virus and the neo gene from transposon Tn5, allowing infected cells to be selected on the basis of G418 resistance. The murine cell lines CTLL-2 and FD.C/1, which are dependent for growth on IL-2 and IL-3, respectively, were both readily infected with the LSNLsrc virus. LSNLsrc-infected, G418-resistant cultures of FD.C/1 cells were able to give rise to IL-3-independent progeny, but all G418-resistant CTLL-2 cells retained normal IL-2 dependence. The induction of IL-3 independence by v-src was not a direct event, since limiting dilution analysis of the LSNLsrc-infected FD.C/1 cells showed that most of them were IL-3 dependent, despite expression of v-src mRNA and active pp60v-src kinase. However, clones selected from this population in the presence of IL-3 were able to undergo a subsequent progression event and generate IL-3-independent progeny. The generation of factor-independent variants in the clonal cultures was a rare event, as witnessed by the death of most of the cells in each clone when IL-3 was withdrawn. Together, these data indicate that a secondary event, in addition to v-src expression, was required to generate IL-3-independent growth. No evidence was found for an autocrine mechanism of transformation involving IL-2, IL-3, interleukin 4, or granulocyte-macrophage colony-stimulating factor.     

The 35-kilodalton protein gene (p35) of Autographa californica nuclear polyhedrosis virus and the neomycin resistance gene provide dominant selection of recombinant baculoviruses.

Autographa californica nuclear polyhedrosis virus (AcMNPV) recombinants were constructed to test the effectiveness of the AcMNPV 35-kilodalton protein gene (35K gene) and the bacterial neomycin resistance gene (neo) as dominant selectable markers for baculoviruses. Insertion of the AcMNPV apoptosis suppressor gene (p35) into the genome of p35-deletion mutants inhibited premature host cell death and increased virus yields up to 1200-fold at low multiplicities in Spodoptera frugiperda (SF21) cell cultures. When placed under control of an early virus promoter, the bacterial neomycin resistance gene (neo) restored multiplication of AcMNPV in the same cells treated with concentrations of the antibiotic G418 that inhibited wild-type virus growth greater than 1000-fold. The selectivity of these dominant markers was compared by serial passage of recombinant virus mixtures. After four passages, the proportion of p35-containing virus increased as much as 2,000,000-fold relative to deletion mutants, whereas the proportion of neo-containing viruses increased 500-fold relative to wild-type virus under G418 selection. The strength and utility of p35 as a selectable marker was further demonstrated by the construction of AcMNPV expression vectors using polyhedrin-based transfer plasmids that contain p35. Recombinant viruses with foreign gene insertions at the polyhedrin locus accounted for 15 to 30% of the transfection progeny. The proportion of desired viruses was increased to greater than 90% by linearizing the parental virus DNA at the intended site of recombination prior to transfection. These results indicate that p35 and neo facilitate the selection of baculovirus recombinants and that p35, in particular, is an effective marker for the generation of AcMNPV expression vectors.