Genomic DNA-mediated gene transfer for argininosuccinate synthetase

Canavanine-resistant (Can^r) human cells overproduce argininosuccinate synthetase without the occurrence of gene amplification. Using calcium phosphate precipitation, genomic DNA from Can^r human cells was used to carry out gene transfer into Chinese hamster cells, which do not express argininosuccinate synthetase activity. Growth in tissue culture medium with citrulline substituted for arginine was adequate to select enzyme-positive colonies. Six independent isolates were selected for detailed analysis by enzyme assay, Southern blotting, Northern blotting, and S1 nuclease analysis, the last of which distinguishes human and hamster mRNA. Five isolates were transferrants containing the human structural gene and synthesizing human enzyme. One isolate represented a cell line synthesizing Chinese hamster enzyme. The data document (1) gene transfer of DNA fragments at least 80 kb in length, (2) the low level of spontaneous activation of the argininosuccnate synthetase locus in Chinese hamster cells, (3) the feasibility of this expression and selection system for DNA-mediated gene transfer, and (4) a method for distinguishing the human and hamster gene products at an RNA level.     

Enhancing the efficiency of DNA-mediated gene transfer in mammalian cells

We have investigated several of the experimental factors that affect calcium phosphate-DNA-mediated gene transfer of thymidine kinase (tk) into mouse LM tk^– Cl 1D cells using unfractionated DNA from both Chinese hamster ovary cells and L6 rat myoblasts. Increases in the length of exposure to DNA (24 h) and the expression time (48 h) before selection result in a 20-fold enhancement in the efficiency of transformation. These modifications yield frequencies up to 35 HAT^R colonies/20 g tk⁺ DNA/10⁶ recipient cells. Exposure to dimethyl sulfoxide enhances transformation efficiencies slightly for short DNA exposure times, but has no effect when optimal DNA exposure times are used. Several other variations in our standard transformation protocol were also examined: these include the concentration and size of the DNA and exposure to low concentrations of the nonionic detergent, Tween-80. We have also isolated and characterized a subclone of Cl 1D that is a high-efficiency recipient for the tk⁺ marker. Segregation analysis reveals that the majority of the Tk⁺ transformants derived from this subclone are stable, in contrast to those derived from the Cl 1D parent. The combination of improved methodology and the highefficiency recipient subclone permits DNA-mediated transformation for tk at frequencies on the order of 10^–4 transformants per recipient cell.     

Molecular cloning of a gene involved in methotrexate uptake by DNA-mediated gene transfer

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from a wild-type Chinese hamster ovary genomic cosmid library. Primary and secondary transfectants, which contain a limited number of cosmid sequences, have been shown to regain methotrexate sensitivity and to take up methotrexate. Furthermore, the DNA from three cosmid clones, isolated from a primary methotrexate-sensitive transfectant, after transfection rescued the methotrexate-resistant phenotype at a high frequency. Restriction endonuclease analysis of the DNA of these cosmid clones indicated that they overlapped extensively and shared two regions of Chinese hamster ovary DNA of 6.6 kb and 20.6 kb. These observations indicate that a gene involved in methotrexate uptake is contained in its entirety within one of these regions. This is the first report of the functional molecular cloning of a gene involved in methotrexate uptake. A general strategy is also described for screening large cosmid libraries from primary transfectants.     

Amphotericin B enhances efficiency of DNA-mediated gene transfer in mammalian cells

Using plasmids containing the genes for thymidine kinase (tk) and neomycin resistance (neo), we have shown that DNA-mediated genotypic transformation of L and Chinese hamster ovary (CHO) cells is increased several-fold by the presence of the sterolbinding polyene antibiotic, amphotericin. Transformation into the same host cells, using genomic DNA, was also enhanced by amphotericin. Phenotypic expression of -galactosidase activity of a plasmid containing the gene for the enzyme was also markedly elevated when the antibiotic was added at transfection. Other sterol-binding polyene antibiotics also showed activity in these DNA-mediated gene transfer assays.     

Identification of human gene complementing ts AlS9 mouse L-cell defect in DNA replication following DNA-mediated gene transfer

The temperature-sensitive (ts) mouse L-cell, ts AlS9, is defective in a gene required for nuclear DNA replication early in the S phase of the cell cycle. Human DNA sequences were introduced into ts AlS9 cells together with the plasmid pSV2neo, which can confer resistance to the drug geneticin. Cotransformants, expressing both the plasmid-derived neomycin gene and the transferred human AlS9 gene, were selected for growth in the presence of the drug at the nonpermissive temperature (npt). The resulting transformants retained a common set of human-specific Alu repetitive DNA sequences. These are likely to be accommodated within, or in proximity to, the transferred human AlS9 gene. The results obtained provide the basis for cloning human genes required for DNA replication.     

DNA-mediated restoration of phenylalanine hydroxylase gene expression in enzyme-deficient derivatives of enzyme-constitutive mouse cell hybrids

Phenylalanine hydroxylase (PH) gene expression is not extinguished in hybrids between PH^– mouse A9 cells, or its neomycin-resistant derivative A9Neo-3, and PH⁺ mouse erythroleukemia (MEL) cells, PHC-3A, in contrast to its extinction in hybrids between A9Neo-3 and PH⁺ rat hepatoma cells, FT-2. Two different types of 6-thioguanine (TG)-resistant derivatives of these A9 x PHC-3A hybrids (LP), are generated in regard toPH gene expression. In regular growth medium supplemented with 10^–4 M TG (Tyr⁺/TG), TG^r derivatives, all of which continue to express PH, occur with high frequency (10^–3). In contrast, in tyrosine-deficient selective medium, supplemented with 10^–4 M TG (Tyr^–/TG), no actively growing colonies are observed. Nevertheless, small colonies containing quiescent cells can be rescued by supplementing the medium with tyrosine. The rescued TG^r clones do not express any detectable level of PH. Biochemical, hybridization, and cybridization analyses of one such rescued clone, LPTG-3, showed that these cells lack the regulatory factor capable of activatingPH gene in PH^– MEL cells. The PH^– phenotype of LPTG-3 cells can be converted to the PH⁺ phenotype by transfection with restriction enzyme-digested or -undigested PHC-3A or mouse liver DNA. Therefore, these cells could be used to clone a fragment of DNA involved inPH gene regulation through DNA-mediated gene transfer methods.     

Liposome-mediated gene transfer and expression via the skin

A ß-galactosidase gene expression construct was usedto investigate the effectiveness of gene delivery and expressionwhen DNA/liposome complexes were topically applied to mouseskin in vivo. DNA was complexed with a commercial preparationof N-[1-(2,3-dioleoyloxy) propyl]-N, N, N-trimethyl-ammonium-methyl-sulphate(DOTAP) in a ratio of 1:1.6 (w/w). The DNA rapidly penetratedthe skin and was expressed in the epidermis, dermis and hairfollicles. A DNA concentration of 267 µg/ml DNA was foundto be optimal for efficient transfection. Expression was seenas early as 6 h post-application, persisted at high levels 24and 48 h post-treatment, but was markedly reduced by 7 daysafter application. In conclusion, utilising a commercially availableliposome preparation, topically-applied DNA/liposome complexescan be efficiently delivered and expressed in several cell typeswithin the skin. This simple, non-invasive technique may haveimplications for a number of gene therapy applications.     

Valproic acid enhances gene expression from viral gene transfer vectors

Viral vectors represent an efficient delivery method for in vitro and in vivo gene transfer, and their utility may be further enhanced through the use of pharmacologic agents that increase gene expression. Here, we demonstrate that valproic acid (VPA), a drug which is widely used for the treatment of epilepsy and mood disorders, enhances and prolongs expression of exogenous genes in cells transduced with various gene transfer agents, including adenovirus, adeno-associated virus and herpesvirus vectors. This effect occurs in a wide range of cell types, including both primary cells and cell lines, and appears to be associated with VPA's ability to function as a histone deacetylase inhibitor (HDACi). VPA treatment also enhanced adenovirally-vectored expression of a luciferase reporter gene in mice, as demonstrated by in vivo imaging. VPA was also less cytotoxic than a commonly used HDAC inhibitor, TSA, suggesting its use as a safer alternative. Taken together, these results suggest that VPA treatment may represent a useful approach to various gene transfer approaches in which enhanced transgene expression is desirable.     

重组乙肝表面抗原真核表达质粒pVAX-S2S的构建及DNA免疫

目的为探索更安全的乙型肝炎病毒(HBV)DNA疫苗,构建编码乙肝表面抗原中蛋白的卡那霉素抗性真核表达质粒,并观察其诱导BALB/c小鼠产生体液免疫应答情况.方法采用限制性内切酶从重组的真核表达质粒pcDNA-S2S中分离出乙肝表面抗原中蛋白(preS2+S)基因片段,将其亚克隆于pVAX1真核表达载体,酶切鉴定,按不同剂量一次性肌内注射免疫小鼠,ELISA法检测小鼠血清抗-HBs.结果酶切鉴定重组质粒pVAX-S2S为正向插入的阳性克隆.HBVDNA疫苗(pVAX-S2S)高(100μg/只)、中(50μg/只)、低(10μg/只)三组剂量一次性免疫健康BALB/c小鼠,均能在2 w诱导抗-HBs产生,抗体效价随时间延长而增长.血清抗体水平比较,高剂量组(97.83±38.78)mU/ml较中剂量组(45.13±21.12)mU/ml、低剂量组(19.74±11.92)mU/ml差异均具显著性(P＜0.05),以后的4,8 w高、中剂量组间差别缩小,但两者较低剂量组差异均具显著性(P＜0.05)和非常显著性(P＜0.01).结论卡那霉素抗性的重组质粒pVAX-S2S能有效诱导正常小鼠产生体液免疫应答.     

Tetracycline-regulated gene expression following direct gene transfer into mouse skeletal muscle

For most experimental and therapeutic applications of gene transfer, regulation of the timing and level of gene expression is preferable to constitutive gene expression. Among the systems that have been developed for pharmacologically controlled gene expression in mammalian cells, the bacterial tetracycline (tet)-responsive system has the advantage that it is dependent on a drug (tet) that is both highly specific and non-toxic. The tet-responsive system has been previously used to modulate expression of cell cycle regulatory proteins in cultured cells, reporter genes in plants and transgenic mice and reporter genes directly injected into the heart. Here we show that orally or parenterally administered tet regulates expression of tet-responsive plasmids injected directly into mouse skeletal muscle. Reporter gene expression was suppressed by two orders of magnitude in the presence of tet, and that suppression was reversed when tet was withdrawn. These data show that skeletal muscle offers an accessible and well characterized target tissue for tet-controlled expression of genesin vivo, suggesting applications to developmental studies and gene therapy.     

Physical mapping of the human cytomegalovirus (HCMV) (Towne) DNA polymerase gene: DNA-mediated transfer of a genetic marker for an HCMV gene

DNA-mediated transfer of a drug resistance marker (phosphonoacetate resistance) for the HCMV (Towne) DNA polymerase (pol) gene has been used to genetically confirm the physical localization of the HCMV (Towne) DNA pol gene. The HCMV (Towne) genomic region homologous to the pol genes of other herpesviruses was first identified by moderate stringency Southern hybridization. Restriction fragments from this region were molecularly cloned from previously characterized phosphonoacetic acid resistant (PAAr) HCMV genomes (R. T. D'Aquilla and W. C. Summers, J. Virol., 61, 1291-1295, 1987). A high frequency of recombinant PAAr virus was found among the progeny of cotransfections of infectious, wild-type HCMV (Towne) DNA only with pol-homologous restriction fragments cloned from PAAr HCMV. The co-transfection technique described here may facilitate further gene mapping in HCMV. The results presented here provide functional proof that the HCMV pol gene is encoded by the sequences previously identified as homologous to other herpesvirus pool genes.     

Efficient DNA-mediated transfer of selectable genes and unselected sequences into differentiated and undifferentiated mouse melanoma clones

We have found that three phenotypically dissimilar mouse B16 melanoma subclones are competent recipients for DNA-mediated gene transfer. Two of these approach and a third, amelanotic clone B78H1, surpasses mouse LTK^– cells in frequencies of transferent colony formation after treatment with either of two codominantly selectable plasmid vectors, pSV2gpt or pGCcos3neo. Melanoma transferents incorporate both selectable plasmid-homologous sequences and substantial amounts of unselected donor DNA into their cellular DNAs. In addition they retain the distinctive states of differentiation characteristic of the untreated clones. Frequencies of pGCcosSneo-mediated transfer of neo gene-encoded antibiotic resistance into B78H1 can reach 10^–2 in response to treatment with as little as 15 ng plasmid/ml coprecipitate/dish. B78H1 cells readily give rise to secondary transferents for the neo gene after treatment with DNA from a primary B78H1 neo transferent. This gene transfer system has potential applications for study of regulation of melanoma and neural crest differentiation and malignancy.     

Retroviral transfer and expression of the interleukin-3 gene in hemopoietic cells

A recombinant retrovirus containing the interleukin-3 (IL3) coding sequence and the neomycin-resistance gene (Neor) has been generated. Infection of fetal liver cells with the IL3 retrovirus, but not with the N2 parental virus, resulted in the formation of factor-independent, NeoR colonies containing various types of differentiated hemopoietic cells. Established cell lines could be generated from these mixed hemopoietic colonies. These cell lines contained the unrearranged viral genome, produced viral IL3, and secreted the growth factor; however, they were not tumorigenic. Identical results were obtained from infection of two factor-dependent cell lines with the IL3 virus, except that these clones all became tumorigenic. These data indicate that endogenous IL3 production can support normal differentiation and immortalization of primary hemopoietic cells, or, in previously immortalized cells, can lead to tumorigenicity.     

Gene expression in implanted rat hepatocytes following retroviral-mediated gene transfer

An hepatocyte transplantation-gene transfer protocol has been developed whereby liver cells containing an expressing Neo^R gene can be successfully implanted in vivo. Adult primary cultures of rat hepatocytes, after infection with the retroviral vector N2, were grown on a floating solid support (coated with purified collagen IV) in a serum-free hormonally defined medium designed for hepatocytes that also contained G418. Under these conditions, normal adult hepatocytes expressing the Neo^R gene could be grown to high density. The solid supports holding the gene-engineered hepatocytes were then implanted into adult rats into subcutaneous and intraperitoneal sites. After one to two weeks, the supports were removed and shown to still contain the gene-engineered hepatocytes expressing the Neo^R gene. These results suggest that cells from solid organs, such as the liver, are potential targets for gene transfer and expression studies in vivo.     

转VEGF基因对血管损伤后修复过程中基质金属蛋白酶表达的影响

目的: 观察局部转染血管内皮生长因子(VEGF)基因对血管球囊拉伤后修复过程中基质金属蛋白酶(MMPs)及其组织抑制因子(TIMPs)表达的影响。方法: 90只新西兰大白兔随机分为3组,Ⅰ组单纯拉伤右髂动脉;Ⅱ组拉伤后局部转染真核表达质粒AdtrackCMV;Ⅲ组拉伤后局部转染pAdtrackCMV-VEGF165;每组按实验终点随机分为5个亚组,术前一周开始给予高脂饮食至实验终点。取拉伤段髂动脉用于总RNA提取、组织病理和免疫组化检测。结果: 3组动物血脂水平保持在正常的5-10倍,组间血管损伤程度评分相似(P＞0.05)。Ⅰ、Ⅱ组整个观察过程中均可检测到MMP2、TIMP1,2持续表达;而转VEGF基因组术后3d时可检测到MMP1,2,9、TIMP1,2表达,1周时达到高峰,8周时无表达。结论: 血管损伤后修复过程中存在MMPs和TIMPs表达失衡,致使基质堆积,并发生病理性重塑,而局部转染VEGF基因选择性改变局部MMPs表达,促进基质降解和适应性重塑。     

Pantropic retroviral vectors mediate gene transfer and expression in Entamoeba histolytica.

Transformation of Entamoeba histolytica has been previously reported, but the foreign genes have all been replicated episomally. Pantropic retroviral vectors based on the Moloney murine leukemia virus with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) have an extremely broad host range and can be concentrated to high titer. To investigate whether these pseudotyped, pantropic vectors can mediate gene transfer and expression in E. histolytica, we constructed a retroviral vector, in which a hygromycin phosphotransferase is expressed from the E. histolytica actin promoter. Data confirm the infection, integration, and expression of a foreign gene mediated by the provirus. To our knowledge, this is the most evolutionarily distant example of successful integration and expression of a mammalian retrovirus. Pantropic retroviral vectors may thus facilitate genetic analysis in species lacking transformation systems.     

Histone-mediated transfer and expression of the HIV-1 tat gene in Jurkat cells

We studied the gene transfer efficiency of lipofection reagents in comparison to DEAE-Dextran. DOTAP, Dosper, and Lipofectin have lower transfection efficiency; Lipofectamine has a 2.5-fold better efficiency compared with DEAE-Dextran. We report a novel and highly efficient DNA transfer system based on the DNA-binding proteins histone 3 and histone 4. We have transferred the HIV-1 tat gene and measured the transactivation of HIV-1 LTR by the transactivator protein, expressed in Jurkat cells. The HIV-1 LTR was linked to the CAT gene as a reporter. Compared to DEAE-Dextran-mediated transfection, histone-mediated transfection resulted in a sevenfold higher expression of the CAT gene. The maximum transfection efficiency mediated by histones is dependent on the relative concentration (DNA:histone ratio) and the incubation time. In a gel-retardation assay, an optimal complex formation was observed under the same conditions that allowed the highest transfection efficiency. This ability of histones to increase the delivery and transgenic expression of foreign DNA in eukaryotic cells is not simply due to the positive ionic character of the histone proteins. Polylysine, histone H1, and histone H2A were unable to mediate gene transfection in our system. Monoclonal antibodies that recognize antigenic determinant present on all five histone proteins (anti-histone, pan) were able to neutralize the transfection-enhancing potential of histone 3 and histone 4. However, anti-histone IgG enhanced the retardation of mobility of histone-DNA complexes. The results of this study allow us to conclude that histones H3 and H4 can catalyze gene transfer and gene expression in eukaryotic cells without any requirement for additional constituents. For this reason, we have termed the new gene-delivery system as histonefection.