Rat embryo fibroblast cells expressing human papillomavirus 1a genes exhibit altered growth properties and tumorigenicity.

Human papillomavirus 1a (HPV1a) induces benign tumors (papillomas or warts) in humans under natural conditions of infection but has not been found to replicate significantly in cell culture or in experimental animals. To establish model systems to study the oncogenic properties and expression of HPV genes, we established cell lines by cotransfecting the 3Y1 rat fibroblast cell line with HPV1a DNA constructs containing an intact early gene region and the Tn5 neomycin resistance gene. Most cell lines selected for expression of the neomycin resistance gene by treatment with the antibiotic G-418 contained viral DNA in a high-molecular-weight form. The growth characteristics of several cell lines containing high copy numbers of HPV1a DNA were studied further. They were shown to differ from the parental cell line and from G-418-resistant cell lines that did not incorporate viral DNA in the following properties: morphological alteration, increased cell density at confluence, growth in 0.5% serum, efficient anchorage-independent growth in soft agar, and rapid formation of tumors in nude mice. Those cell lines that possessed altered growth properties and tumorigenicity were found to express abundant quantities of polyadenylated virus-specific RNA species in the cytoplasm.     

Characterisation of G418-induced metabolic load in recombinant CHO and BHK cells: effect on the activity and expression of central metabolic enzymes

In a previous article (Yallop and Svendsen 2001), recombinant CHO and BHK cell lines, expressing the human glucagon receptor and the gastric inhibitory peptide receptor, respectively, showed reduced growth rates and altered nutrient utilisation when grown with increasing concentrations of G418. This response was associated with an increased expression of the neo ^r protein, while expression of the recombinant membrane receptors remained unaltered. The metabolic response was characterised in both cell lines by an increase in the specific rate of glutamine utilisation and in CHO cells by a decrease in the yield of lactate from glucose, suggesting a change in the flux of glucose through central metabolism. The aim of this study was to further elucidate these metabolic changes by determining the activity and relative expression of key enzymes involved in glucose and glutamine metabolism. For both CHO and BHK cells, there was an increase in the activity of glutaminase, glutamate dehydrogenase and glutamine synthetase, suggesting an increased flux through the glutaminolysis pathway. The activity of glucose-6-phosphate dehydrogenase and pyruvate carboxylase in CHO cells was also increased whilst lactate dehydrogenase activity remained unaltered, suggesting an increased flux to the pentose phosphate pathway and TCA cycle, respectively. The activity of these enzymes in BHK cells was unchanged. Quantitative RT-PCR showed that expression levels of glutaminase and pyruvate carboxylase were the same with and without G418, indicating that the differences in activities were likely due to post-translational modifications. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

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.     

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.     

High efficiency gene transfer into murine T cell clones using a retroviral vector

To establish a gene transfer and expression system for murine T cell clones, we have introduced the neomycin phosphotransferase gene encoding resistance to the neomycin analogue, G418, into non-neoplastic inducer T cell clones by using a replication-defective retroviral vector. This method allowed highly efficient gene transfer (20 to 40%) into two inducer T cell clones. The level of viral RNA expression in G418r T cells was 0.1% of poly(A)+ RNA. The infected G418r cells retained physiologic responsiveness to specific antigen as judged by antigen-specific proliferation and production of IL 3.     

The Production of a Stably Transformed Insect Cell Line Expressing An Invertebrate GABAA Receptor β-Subunit

Abstract

We have produced a stable insect cell line derived from Spodoptera frugiperda (Sf9) cells expressing a cDNA encoding a β-subunit of the Lymnaea stagnalis GABA_A receptor. The cDNA was randomly integrated into the insect cell genome under the control of a baculovirus immediate early gene (IE-1) promoter. Stable cell lines were established by transformation of Sf9 cells with the expression vector pIEK1.LGβ1 together with a plasmid encoding a selectable marker which confers neomycin (G418) resistance. Following growth in the presence of G418, neomycin resistant clones were selected, amplified and analysed for the presence of functional GABA-gated chloride channels. Electrophysiological analysis of one cell line showed the presence of a picrotoxin-sensitive chloride channel not present in control Sf9 cells. These channels were also sensitive to GABA, albeit at relatively high (mM) concentrations.     

Enhanced sensitivity to G418 of human KB cells adapted to certain media and sera.

Sensitivity to the neomycin derivative G418 was determined for a human cell line, KB 3-1, that had been adapted to six different combinations of media and sera. The results indicate that while the plating efficiency is similar for all conditions, the susceptibility to G418 can differ markedly depending on the particular combination of media and sera used. This suggests that in experiments using neomycin resistance as a selectable marker, conditions may be found where the amount of G418 required for selection and maintenance of transfected cell lines can be reduced, providing a significant savings.     

Gene activation mediated by protein kinase C in human macrophage and teratocarcinoma cells expressing aminoglycoside phosphotransferase activity.

The bacterial neomycin phosphotransferase gene driven by the Moloney mouse leukemia virus long terminal repeat (LTR) or SV40 early region promoter was introduced into the human promonocyte-macrophage cell line, U937, and into the pluripotential human embryonic teratocarcinoma cell line, NT2/D1. Clonally derived cell lines capable of growing in 2-4 mg/ml of the aminoglycoside antibiotic, G418 (Geneticin), were established and transfected with pHIVCat, a plasmid expressing the bacterial chloramphenicol acetyl transferase (CAT) activity under the control of the human immunodeficiency virus (HIV-1) LTR. All of the G418 resistant (neo(r)) U937 cell lines and 10 of 14 neo(r) NT2/D1 cell lines exhibited reduced basal levels of CAT expression or impaired responses to activation of the HIV-1 LTR by phorbol 12-myristate 13-acetate (PMA) when compared to the parental lines. Other differences included inhibition of tat activation of the HIV-1 LTR and increased sensitivity of U937 cells to human tumor necrosis factor alpha. The expression of other eukaryotic promoters including the HTLV-1 LTR, SV40 ori sequences, and the human beta-actin gene promoter was similarly affected. However, differentiation of the neo(r) U937 cells into macrophages was neither delayed nor impaired. Because PMA is an activator of protein kinase C (PKC) and a potent inducer of HIV-1 directed gene expression, the amounts, sensitivity to G418, and cytosol to membrane translocation of this enzyme were determined in the wild type and neo(r) U937 cells. G418 at concentrations too low to affect cell growth (12-150 micrograms/ml) inhibited PMA-induced transactivation responses in wild type cells but did not inhibit PKC-dependent protein phosphorylation in vitro. PKC activities in the wild type and neo(r) cells were similar in absolute amounts and in the cytosol-membrane distribution of the enzyme. In contrast with wild type cells, however, all of the cytosolic Ca(2+)-phospholipid-dependent form of PKC disappeared from the neo(r) cells within 30 min after PMA induction. The results suggested that, depending upon the cell type, gene cotransfer using aminoglycoside resistance as a selectable marker may seriously perturb important cellular control mechanisms such as the PKC pathway leading to activation of gene expression.     

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．     

Effects of exogenous wild-type p53 on a human lung carcinoma cell line with endogenous wild-type p53

Several studies have shown that expression of exogenous wild-type p53 is detrimental to the growth of cell lines with absent or mutant p53. In this study, wild-type p53 cDNA expression plasmids were transfected into A549 lung carcinoma cells which had previously been shown by sequencing to contain wild-type p53. When a constitutively expressed wild-type p53 plasmid containing the neomycin resistance gene was transfected into these cells, no G418-resistant colonies contained the exogenous p53 cDNA even though the neomycin resistance gene was integrated. When cells were transfected with a dexamethasone-inducible wild-type p53 cDNA expression plasmid, induction of p53 expression resulted in a decreased growth rate and a decreased proportion of S-phase cells. Continuous treatment with dexamethasone resulted in continued p53 expression for 16 days, but beyond that time expression ceased and could not be reinduced. These data indicated that although the A549 cell line could proliferate in the presence of endogenous wild-type p53 there was a strong selection pressure against continued expression of additional exogenous wild-type p53.     

Metabolic burden in recombinant CHO cells: effect ofdhfr gene amplification andlacZ expression

Foreign protein production levels in two recombinant Chinese hamster ovary (CHO) cell lines were compared in cells transfected with different expression vectors. One vector pNL1 contained the gene for neomycin resistance (neo ^r ) and thelacZ gene which codes for intracellular -galactosidase, with both genes controlled by the constitutive simian virus (SV40) promoter. The other vector CDG contained the amplifiabledhfr gene andlacZ gene, controlled by the constitutive SV40 and cytomegalovirus (CMV) promoters, respectively. Cell growth and -galactosidase expression were compared quantitatively after cells were selected in different concentrations of the neomycin analog G418 and methotrexate, respectively. A 62% reduction in growth rate occurred in recombinant CHO cells in which thelacZ anddhfr genes were highly amplified and expressed. In contrast, the combined effects of the unamplifiedneo ^r gene andlacZ gene expression on the growth kinetics were small. Any metabolic burden caused bylacZ gene expression, which was evaluated separately from the effect ofneo ^r gene expression, must be negligible, as higher expression of -galactosidase (1.5×10^–6 units/cell) occurred in unamplified cells compared to the cells in whichlacZ was amplified by thedhfr-containing vector (3×10^–7 units/cell). Thus, the main factor causing severe growth reduction (metabolic burden) in cells containing the amplifieddhfr gene system was not overexpression of -galactosidase butdhfr andlacZ gene co-amplification anddhfr gene expression.     

A novel G418 conjugate results in targeted selection of genetically protected hepatocytes without bystander toxicity

G418, an aminoglycoside neomycin analogue, is an antimicrobial agent that interferes with protein synthesis and has been used extensively for selection of mammalian cell lines that possess neomycin resistance (NR). It is potent and nonspecific in its effects that occur through tight binding to ribosomal elements. Because of the potent intracellular effect, we wondered whether G418 could be used to select a specific cell type based on receptor-mediated endocytosis. The objective of this study was to target G418 specifically to liver cells via asialoglycoprotein receptors (AsGR) which are known to be highly selective for these cells. A novel G418 conjugate was synthesized chemically by coupling G418 to a galactose-terminating carrier protein, asialoorosomucoid (AsOR), in a molar ratio of 5:1. AsOR-G418 conjugates inhibited viability of AsGR (+) cells by 84.3%, while inhibition in AsGR (-) cells was only by 19%. In AsGR (+) cells, stably transfected with a NR gene, the conjugate decreased viability by less than 9%. Furthermore, incubation of conjugate in cocultures of AsGR (+), and AsGR (-) cells did not result in the loss of viability of neighboring AsGR (-) cells. Our data demonstrate for the first time that G418 can be covalently bound to AsOR to form a conjugate for hepatocyte-specific targeting and toxicity. AsOR-G418 conjugates may be useful tools for genetic manipulation of human liver cells in the presence of nonhepatic cells.     

In vitro synthesis of infectious retroviral RNA.

A plasmid was constructed in which a T7 RNA polymerase promoter was placed upstream of a recombinant amphotropic retrovirus genome containing a selectable neomycin resistance gene. To test the infectivity of the RNA produced by T7 RNA polymerase in vitro, the RNA was microinjected into the nuclei of psi 2 packaging cells. Infectious particles conferring G418 resistance were released.     

A transformation system for the hypotrichous ciliate Stylonychia mytilus

We describe a transformation system for the ciliate Stylonychia mytilus. The neomycin resistance gene from Escherichia coli transposon Tn5, which codes for the enzyme phosphotransferase and confers resistance to the antibiotic G 418, was ligated into macronuclear `gene-size' DNA molecules. Using this recombinant DNA for transformation experiments we show that the gene is replicated and expressed in transformed cells.     

Construction of a recombinant bovine leukemia virus vector for analysis of virus infectivity.

A recombinant bovine leukemia virus (BLV) was constructed in which the X region was replaced with the bacterial neomycin resistance gene controlled by the simian virus 40 early promoter. This virus, termed BLV-SVNEO, is a self-packaging, activator-dependent retroviral vector. Introduction of the plasmid pBLV-SVNEO into mammalian cells resulted in constitutive expression of the neo gene, whereas the BLV structural genes, gag, pol, and env, were expressed only in the presence of the two regulatory proteins, Tax and Rex. The production and release of recombinant virus by cells transfected with pBLV-SVNEO were proportional to the number of G418-resistant colonies that developed after susceptible cells were exposed to the filtered culture medium. BLV-SVNEO was able to infect cell lines of human, bovine, canine, feline, and murine origin. BLV-producing cell lines were resistant to superinfection with BLV-SVNEO. This cell-virus system should facilitate molecular genetic studies of BLV and will provide a rapid, quantitative measure of BLV infectivity in a variety of cell types. These studies also demonstrate the feasibility of using activator-dependent retroviral vectors such as BLV-SVNEO to deliver foreign genes into cells and eventually animals.     

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.     

Protection of uninfected human bone marrow cells in long-term culture from G418 toxicity after retroviral-mediated transfer of the NEOr gene

The long-term effect of retroviral-mediated gene transfer into human hematopoietic cells in vitro was studied in bone marrow culture. Two retroviral vectors (pN2 or pZIP NEO) were used to transfer the gene coding for neomycin phosphotransferase, which confers neomycin resistance, as a dominant selectable marker. Following infection, bone marrow cells of multiple hematopoietic lineages displayed resistance for the duration of the cultures (greater than 80 days) to normally cytotoxic doses of the neomycin analog G418. However, upon DNA analysis of cells surviving in G418, the NEOr (neomycin resistance) gene was not detected under conditions where single copy genes could readily be seen, despite the presence of NEOr RNA sequences. In order to investigate this observation further, infected and uninfected cells were separated by a filter, and cultured in the same medium containing G418. The uninfected cells continued to survive in the presence of normally toxic concentrations of G418. Medium alone from infected cells was able to protect uninfected cells the same way. Efficiency of transfer of this and perhaps other selectable marker genes to cells in the long-term culture system may consequently be overestimated if survival of cells alone is quantitated. These results indicate that long-term cultures are a useful in vitro model for the study of retroviral-mediated gene transfer to human hematopoietic cells.