Plasmidial maintenance in rodent fibroblasts of a BPV1-pBR322 shuttle vector without immediately apparent oncogenic transformation of the recipient cells.

A recombinant plasmid was constructed (pV69) which comprises a subgenomic fragment of bovine papilloma virus type 1 (BPV1) DNA, part of plasmid pBR322 DNA and a drug resistance gene expressed in both mammalian fibroblasts and Escherichia coli. This gene (vv2) is a modified form of the bacterial neomycin resistance gene (neo) linked to the herpes simplex virus thymidine kinase (tk) promoter (plasmid pAG60), to which the original bacterial neo promoter from transposon Tn5 was added back, upstream of the eukaryotic promoter. It induced kanamycin resistance in E. coli, as well as resistance to the drug G418 in rat and mouse fibroblasts. Its expression in FR3T3 rat cells was enhanced as compared with the original tk-neo construction. After transfer of plasmid pV69 into C127 mouse cells or FR3T3 rat cells, the number of resistant colonies selected in medium containing G418 was one to two orders of magnitude higher than that of transformed foci in normal medium. In eight independent cell lines selected by drug resistance, pV69 DNA was found to be maintained in a plasmidial state, without any detectable rearrangement or deletion and could be transferred back in E. coli. In contrast, cell lines selected by focus formation in normal medium maintained deleted forms of the original plasmid DNA, and only part of them were resistant to G418. Most of the drug-resistant clones had kept the morphology and growth control of the normal fibroblasts. However, with further passages in culture, these cells spontaneously produced transformed foci with increasing frequencies.     

Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants.

The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.     

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.     

Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis.

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells.     

Reactivation of thyroglobulin gene expression in transformed thyroid cells by 5-azacytidine

Transformed rat thyroid cells fail to express thyroglobulin. Cells transformed with a Kirsten murine sarcoma virus carrying a temperature-sensitive ras allele lose their transformation phenotype when shifted to the nonpermissive (39 degrees C) temperature. The thyroglobulin promoter, however, remains inactive. Similarly, transfection of these cells with a thyroglobulin promoter fused to a neomycin resistance reporter gene does not produce clones resistant to G418. Treatment of the transfected cells with the DNA demethylating agent 5-azacytidine reactivates the thyroglobulin promoter and yields stable G418-resistant clones. We show that thyroglobulin promoter activity is correlated with the presence of a thyroid-specific nuclear factor, TgTF1. TgTF1 cannot be detected in transformed cells but reappears after treatment with 5-azacytidine at 39 degrees C. Restoration of Ras activity at 33 degrees C leads to the rapid loss of TgTF1 and G418 resistance.     

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。     

Stable transfection of Acanthamoeba castellanii

A simple method for stable transfection of Acanthamoeba castellanii using plasmids which confer resistance to neomycin G418 is described. Expression of neomycin phosphotransferase is driven by the Acanthamoeba TBP gene promoter, and can be monitored by cell growth in the presence of neomycin G418 or by Western blot analysis. Transfected cells can be passaged in the same manner as control cells and can be induced to differentiate into cysts, in which form they maintain resistance to neomycin G418 for at least several weeks, although expression of neomycin phosphotransferase is repressed during encystment. Expression of EGFP or an HA-tagged EGFP-TBP fusion can be driven from the same plasmid, using an additional copy of the Acanthamoeba TBP gene promoter or a deletion mutant. The TBP-EGFP fusion is localized to the nucleus, except in a small proportion of presumptive pre-mitotic cells. EGFP expression can also be driven by the cyst-specific CSP21 gene promoter, which is completely repressed in growing cells but strongly induced in differentiating cells. Transfected cells maintain their phenotype for several weeks, even in the absence of neomycin G418, suggesting that transfected genes are stably integrated within the genome. These results demonstrate the utility of the neomycin resistance based plasmids for stable transfection of Acanthamoeba, and may assist a number of investigations.     

Regulated replication of an episomal simian virus 40 origin plasmid in COS7 cells.

The replication of a simian virus 40 (SV40) origin-containing plasmid, pSLneo, stably transfected COS7 cells has been studied. pSLneo contains the SV40 origin of replication and encodes the positive selectable marker for G418 resistance. In transient replication assays, pSLneo replicates to a high copy number in COS7 cells. Uncontrolled SV40 plasmid replication has been reported to be lethal to such transfected cells. Thus, it was anticipated that extensive plasmid replication would preclude isolation of permanent cell lines containing pSLneo. However, significant number of G418-resistant colonies arose after transfection of COS7 cells with pSLneo. Cell lines established from these drug-resistant colonies contained between 100 and 1,000 extrachromosomal pSLneo copies per cell. Episomal plasmid DNA in pSLneo/COS7 lines was stably maintained after 2 months of continuous culture in selective medium. Bromodeoxyuridine labeling and density shift experiments demonstrated that replication of pSLneo closely paralleled that of cellular DNA. On average, plasmid DNA did not replicate more than once during a single cell generation period. Regulation of pSLneo replication appeared to be negatively controlled by a cis-acting mechanism. Endogenous copies of episomal pSLneo remained at a stable low copy number during the simultaneous, high-level replication of a newly transfected plasmid encoding SV40 large T antigen in the same cells. These results indicate that regulated replication of an SV40 origin plasmid can be acquired in a cell and does not require the presence of additional genetic elements. The molecular mechanism by which cells enforce this regulation on extrachromosomal SV40 plasmids remains to be defined.     

A single locus on human chromosome 21 directs the expression of a receptor for adenovirus type 2 in mouse A9 cells.

The receptors on human cells which mediate adsorption of adenoviruses have not been identified. We found that murine A9 cells and Chinese hamster ovary (CHO) cells failed to bind significant levels of radiolabeled adenovirus type 2 (Ad2) virions but that derivatives of these cells carrying human chromosome 21 exhibited high levels of virus binding that was specific for the viral fiber protein. G418-resistant A9 cell transformants expressing Ad2 receptors were detected at a frequency of about 10(-4) following cotransfection with high-molecular-weight DNAs from mouse cells containing human chromosome 21 and plasmid DNA containing a neomycin resistance gene. The Ad2 receptors on the transformed A9 cells were similar to those on human cells with respect to their concentration on the cell membrane, their affinity for the viral fiber protein, and their ability to direct virus into cells along a pathway leading to delivery of the viral DNA genome into the cell nucleus. Furthermore, identical human DNA fragments were present in three independent mouse cell transformants expressing Ad2 receptors, supporting the conclusion that these human DNA fragments correspond to a gene or locus on chromosome 21 that directs the expression of Ad2 receptors in these cells.     

Recovery of hormonal regulation in protein kinase defective adrenal cells through DNA-mediated gene transfer

A cAMP-resistant mutant (Kin-8) isolated from Y1 mouse adrenocortical tumor cells harbors a specific lesion in the regulatory subunit of the type 1 cAMP-dependent protein kinase. This mutant also is resistant to the effects of corticotropin and cAMP on steroidogenesis, growth and morphology, suggesting an obligatory role for the protein kinase in regulation of adrenocortical functions. In this study, the cAMP-resistant phenotype of the Kin-8 mutant was reverted by transformation with DNA from cAMP-responsive Y1 cells, and the biochemical basis of the transformation was explored. Initially, Y1 mouse adrenocortical tumor cells were evaluated for their competence as recipients in DNA-mediated transformation experiments, by measuring their ability to incorporate and express a bacterial gene (neo) encoding resistance to neomycin. Y1 cells were transfected with the plasmid pSV2-neo (an SV40-neo hybrid vector designed for expression in animal cells) and screened for resistance to the neomycin analog, G418. Neomycin-resistant transformants were recovered from Y1 cells at a frequency of approximately one per 10(3) cells per 10 micrograms of DNA, and had specific neo sequences integrated into their high molecular weight (mw) DNA. The Y1 mutant, Kin-8, then was transformed with pSV2-neo DNA plus high mw DNA prepared from cAMP-responsive Y1 cells. Cells competent for transformation were recovered by selective growth in the neomycin analog G418, and these transformants were screened for recovery of morphological responses to cAMP. Several colonies capable of rounding up in the presence of cAMP were recovered after transformation with DNA from Y1 cells. These transformants also recovered the ability to round up in the presence of corticotropin, and were able to respond to both corticotropin and cAMP with increased steroidogenesis. Transformants generated from either Y1 or Kin-8 cells were unstable. Y1 cells lost resistance to neomycin when grown in the absence of G418 at a frequency of 4% per generation. Similarly, Kin-8 transformants lost their sensitivity to cAMP in subsequent culture passages. In some of the cAMP-responsive transformants, cAMP-dependent protein kinase activity was recovered and approached the activity seen in cAMP-responsive Y1 cells. The recovery of a normal protein kinase by transformation appeared to have been sufficient to reverse the cAMP-resistant phenotype of Kin-8 cells. In other cAMP-responsive transformants, protein kinase activity was not appreciably affected by cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication

Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination.     

Retransformation of a revertant cell line with the adenovirus E1 oncogenes and vanadate

We have isolated a revertant cell line (G5) from an adenovirus transformed rat cell line (F4) which failed to express the integrated viral oncogenes. To determine whether the reversion mutation was acting in cis or trans the G5 cells were co-transfected with an E1 gene bearing expression plasmid and a neomycin phosphotransferase bearing plasmid. G418-resistant colonies were picked and shown to express the E1 proteins and to be tumorigenic. This re-transformation could be partially mimicked by treatment with vanadate, an inhibitor of phosphotyrosine phosphatases. These results show that the continued presence of the E1 proteins was required to maintain the transformed phenotype, and that the reversion mutation was a cis-acting event affecting directly the integrated E1 genes.     

Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei.

Heteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells.     

An immortalized xeroderma pigmentosum, group C, cell line which replicates SV40 shuttle vectors

We have established and characterized an immortalized xeroderma pigmentosum (XP), group C, cell line. Transformation of the human fibroblasts was carried out with a recombinant plasmid, pLAS-wt, containing SV40 DNA encompassing the entire early region with a defective origin of DNA replication. The transformed XP cell line, XP4PA-SVwt, and the normal transformed fibroblasts AS3-SVwt, both express SV40 T antigen together with enhanced levels of the transformation-associated cellular protein, p53. XP4PA-SVwt retains the XP UV-repair defective phenotype as demonstrated by low levels of unscheduled DNA synthesis and by the reduced survival of irradiated SV40 virus. Analysis of cellular DNA shows a single major, stable, integration site of pLAS-wt in the XP4PA-SVwt cells. The T antigen in these cells supports efficiently the replication of SV40 based shuttle vectors and should prove suitable for the introduction, expression and selection of genes related to DNA repair and to the study of mutagenesis using defined molecular probes.     

The bryophyte Physcomitrella patens replicates extrachromosomal transgenic elements

Physcomitrella patens , recently renamed Aphanoregma patens , has been transformed with the plasmid, pBI426. On selective medium approx. 30% of regenerants expressed the transformed phenotype transiently (transients). The remaining 70% (transformants) retained their transformed phenotype (GUS-positive and resistant to G418) indefinitely when subcultured repeatedly on selective medium. However, most lost this phenotype after one or two passages through nonselective medium (unstable transformants). Approximately 0.2% of transformants retained their transformed phenotype after numerous passages through nonselective medium (stable transformants). Using PCR methodology, it has been shown that loss of the transformed phenotype by unstable transformants is invariably accompanied by disappearance of the transgenic DNA. Southern blot analysis data argue strongly that unstable transformants cultured under selective conditions contain unintegrated pBI426 as circular concatenates consisting of 3–40 copies of the plasmid. Under selective conditions, it appears that replication and/or partitioning of these extrachromosomal concatemers might be growth rate-limiting. This is the first report of a transgenic, autonomously replicating extrachromosomal element in a photosynthetic plant. A single copy of pBI426 has been inserted into the moss genome in each of three stable transformants analysed.     

在哺乳动物细胞中稳定表达丙型肝炎病毒E2糖蛋白

利用ＤＮＡ重组技术,将Ⅲ型中国株ＨＣＶＥ２／ＮＳ１基因片段插入真核表达载体,然后转染哺乳动物细胞ＮＩＨ３Ｔ３以表达Ｅ２糖蛋白．检测显示来自３月以上培养的细胞克隆中表达产物分子量为７０ｋＤ,经Ｗｅｓｔｅｒｎｂｌｏｔ证实该表达产物能与抗ＨＣＶ阳性血清进行特异性反应．以上表明首次在哺乳动物细胞中成功表达Ⅲ型中国株ＨＣＶ的Ｅ２糖蛋白,并建立相应的稳定表达细胞系．     

Human papillomavirus type 6b DNA required for initiation but not maintenance of transformation of C127 mouse cells.

We describe the transformation of C127 mouse fibroblasts with human papillomavirus type 6b (HPV-6b) DNA, which is associated primarily with benign tumors of the human genital tract. The major transformed phenotype of the HPV-6b-transfected cells lines, which had been G418 selected, pooled, and maintained without subsequent selection, was tumorigenicity in nude mice. We found that, unlike that reported for other HPVs or papovaviruses, the transformed phenotype was expressed after a delay, in which the cells had undergone extensive culture passages (about 20 passages or 100 generations). Interestingly, the HPV-6b DNA had become reduced or nondetectable in copy number in the cells by the time the transformed phenotype was expressed and in most of the tumors induced by the cells in nude mice, indicating that high levels of HPV-6b DNA were not required for maintenance of the transformed phenotype. Clonal cell lines gave similar results. When continued G418 selection was used to maintain high-copy-number HPV-6b DNA, the cells were tumorigenic, indicating that high levels of HPV-6b DNA did not suppress tumorigenesis. These studies suggest that HPV-6b DNA initiates transformation of C127 cells but is dispensable for expression or maintenance of the transformed phenotype. Transformation by HPV-6b DNA in vitro may provide insights into the HPV type-specific association with benign versus malignant lesions in vivo and may elucidate some of the oncogenic processes involved in tumor progression.