The prokaryotic neomycin-resistance-encoding gene acts as a transcriptional silencer in eukaryotic cells

The gene encoding neomycin resistance (neo) mediates a cis-acting negative effect on expression from promoters in transient and stable transfectants of mammalian cell lines. Inserting the neo gene either into retroviral vectors or into plasmids containing reporter genes results in a five- to tenfold decrease of expression from proximal promoters like the simian virus 40 early or the retroviral myeloproliferative sarcoma virus promoter. The silencing effect is not dependent on the insertion site or the orientation of the fragment. The neo gene is frequently used in eukaryotic vectors as a dominant selectable gene. Other selectable genes were tested for potential cis-activity. We found that the gene conferring resistance to puromycin from Streptomyces alboniger does not influence adjacent promoters.     

Tandem gene amplification in vitro for rapid and efficient expression in animal cells

Plasmid vectors pHSG293 and pHSG747, suitable for in vitro gene amplification for subsequent animal-cell expression, were developed. A cosmid vector pHSG293 confers Km resistance to Escherichia coli host cells and G418 resistance to animal cells and contains a single BstXI recognition/cleavage site, CCACGGGG/CTGG, near the cos site (the recognition site is underlined). The cassette vector plasmid pHSG747 contains a multiple cloning site (MCS) between the simian virus 40 early promoter and the poly(A) signal sequence flanked by the same BstXI sites and confers Cm resistance to E. coli host cells. After inserting a coding fragment for human protein C or its derivative in the appropriate orientation in the MCS of pHSG747, the BstXI expression unit fragment was purified, mixed with BstXI-digested pHSG293 DNA at a molecular ratio of 20 to 40:1 and ligated. This allowed for tandem gene amplification due to asymmetric cohesive ends. Ligation products were packaged in lambda phage particles, amplified in E. coli cells as large cosmid molecules, and then introduced into CHO cells. G418R transformants were found to produce and secrete recombinant protein molecules at a high level. The plasmid vectors developed in this work will provide a rapid screening system useful for protein engineering in animal cells.     

A mouse hybrid cell line that supports gene expression from a variety of promoters in amplifiable vectors

Summary A hybrid cell line was constructed by fusion of mouse L-cells with an NIH3T3 cell line derivative containing a hybrid gene consisting of the mouse immunoglobulin kappa (IgK) variable gene promoter linked to theEscherichia coli gpt gene. Such hybrids grew to a much higher density compared to either of the parental cell lines. The utility of this cell line as a host to express foreign genes was tested by the expression of TGF-β cDNA using the cytomegalovirus promoter. The vector also contained the human dihydrofolate reductase (DHFR) gene driven by SV40 early promoter, to allow for the amplification of the transfected gene. Initial transformants, selected at 100 nM methotrexate (MTX), were subsequently selected for resistance to a higher concentration of MTX (2 μM). Such clones expressed an increased level of TGF-β when compared to the initial transformants. Both the initial transformants and the clones with the amplified DHFR gene produced TGF-β in an acid-activatable precursor form. This mouse hybrid host cell line also allowed the expression of foreign genes cloned in an eukaryotic expression vector with the mouse IgK variable region promoter and human growth hormone as the reporter gene, whereas such vectors did not function in CHO cells. The mouse hybrid cell line was also found to be capable of being used with a broad range of promoters.     

Organization of a Chinese hamster ovary dihydrofolate reductase gene identified by phenotypic rescue.

We have constructed a genomic DNA library from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) in the cosmid vector pHC79. By utilizing a murine dihydrofolate reductase (DHFR) cDNA clone, we have identified 66 DHFR+ clones among the 11,000 colonies screened by colony hybridization. To isolate a recombinant cosmid containing the entire DHFR gene, we have tested these colonies for their ability to rescue a DHFR- Chinese hamster ovary cell line, using the spheroplast fusion method of gene transfer developed by W. Schaffner (Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980). One clone (cH1) was able to transform DHFR- cells to the DHFR+ phenotype and was shown in hybridization studies to contain all of the gene except a small portion of the 3' untranslated region. We have mapped cosmid cH1 and several overlapping cosmids with a variety of restriction enzymes and have determined the approximate positions of the five (and possibly six) exons within the DHFR gene. Differences between the sizes of homologous genes in hamster cells (24.5 kilobases [kb]) and in mouse cells (31.5 kb) are shown to reside primarily in the length of the 3' intron, which is 8 kb in the hamster gene and 16 kb in length in the mouse gene. Our studies confirm the utility of cosmid libraries for the isolation of large genes, as previously shown by R. de Saint Vincent et al. (Cell 27:267-277, 1981). In addition, a cosmid that contains a functional DHFR gene will be a useful vector for the co-amplification and subsequent overexpression of other cloned genes.     

High efficiency vectors for cosmid microcloning and genomic analysis

We describe the construction and use of cosmid vectors designed for microcloning, gene isolation and genomic mapping starting from submicrogram amounts of eukaryotic DNA. These vectors contain (1) multiple cos sites to allow for simple and efficient cloning using non size-selected DNA; (2) bacteriophage T3 and T7 promoter sequences flanking the cloning site to allow for the synthesis of end-specific probes for chromosome walking; (3) a selectable gene for immediate gene transfer of cosmid DNA into mammalian cells; (4) recognition sequences for specific oligodeoxyribonucleotides to allow rapid restriction mapping; (5) unique NotI, SacII or SfiI sites flanking the cloning site to allow for removal of the cloned DNA insert from the vector. These cosmid vectors allow the construction of high quality genomic libraries in situations where the quantity of purified DNA is extremely limited, such as when using DNA prepared from purified mammalian chromosomes isolated by fluorescence-activated cell sorting.     

The establishment of IL-2 producing cells by genetic engineering

Expression plasmids containing human interleukin-2(IL-2) cDNA under the control of viral promoters (SV40 early region, MuLV LTR, HTLV-I LTR, and ASV (Y73) LTR) were introduced into TK- mouse L cells and human FL cells to establish IL-2 producing cells. The highest levels of IL-2 producing clones were obtained in TK+ mouse L cells transformed with a recombinant plasmid having MuLV LTR as a promoter, whereas transformed cells of human FL cells (G418r) were revealed to produce IL-2 at the highest level when the cells were transfected with a plasmid containing HTLV LTR as a promoter. These results suggest that these promoter/enhancer regions possess different cell specificities in gene expression. To obtain higher levels of IL-2 production using gene amplification, the hybrid plasmids containing the hamster DHFR and human IL-2 genes were constructed and transfected into DHFR- CHO cells. DHFR+ colonies produced IL-2 at about the same level as that produced by TK+ L cells transformed with the recombinants containing MuLV LTR. Selection of methotrexate-resistant cells resulted in a 5- to 30-fold increase of IL-2 production. These cells produced IL-2 stably for at least 3 months, even in the absence of methotrexate.     

Conversion through homologous recombination of the gene encoding Simian virus 40 115,000-molecular-weight super T antigen to a gene encoding a normal-size large T antigen variant.

We have previously cloned the gene encoding a 115,000-Mr super T antigen (115K super T antigen), an elongated form of the Simian virus 40 large T antigen, originating from the rat cell line V 11 F1 clone 1, subclone 7 (May et al., J. Virol. 45:901-913, 1983). DNA sequence analysis has shown that the 115K super T antigen gene contains notably an in-phase duplication of a sequence located in the region of tsA mutations. We have also shown that the 115K super T antigen gene is able to induce the formation of transformed foci in transfected rat cells. After rat cell cultures were transfected with the cloned gene encoding 115K super T antigen, we obtained a large number of transformants as reported in this paper. In these transformants, we detected a very high frequency of new T antigen variants, as shown by immunoprecipitation of the cell extracts with anti-simian virus 40 tumor serum followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Based on these results and all of the data presently available, it appears likely that the input plasmid or cosmid DNAs containing the cloned gene were first subjected to recombination events that yield new variant T antigen genes before these recombinant genes become integrated. The new variant T antigens observed in the transformants were predominantly those comigrating with normal-size large T antigen. In fact, these latter variants appeared to be indistinguishable from wild-type large T antigen as judged by restriction mapping by Southern blotting of the total genomic DNA of the transformants. Models of intermolecular or intramolecular homologous recombination occurring between or within the input plasmid or input cosmid DNA molecules are proposed to account for the formation of such revertants.     

Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus long terminal repeat.

We have constructed a recombinant simian virus 40 (SV40) DNA containing a copy of the Harvey murine sarcoma virus long terminal repeat (LTR). This recombinant viral DNA was converted into an infectious SV40 virus particle and subsequently infected into NIH 3T3 cells (either uninfected or previously infected with Moloney leukemia virus). We found that this hybrid virus, SVLTR1, transforms cells with 10 to 20 times the efficiency of SV40 wild type. Southern blot analysis of these transformed cell genomic DNAs revealed that simple integration of the viral DNA within the retrovirus LTR cannot account for the enhanced transformation of the recombinant virus. A restriction fragment derived from the SVLTR-1 virus which contains an intact LTR was readily identified in a majority of the transformed cell DNAs. These results suggest that the LTR fragment which contains the attachment sites and flanking sequences for the proviral DNA duplex may be insufficient by itself to facilitate correct retrovirus integration and that some other functional element of the LTR is responsible for the increased transformation potential of this virus. We have found that a complete copy of the Harvey murine sarcoma virus LTR linked to well-defined structural genes lacking their own promoters (SV40 early region, thymidine kinase, and G418 resistance) can be effectively used to promote marker gene expression. To determine which element of the LTR served to enhance the biological activity of the recombinant virus described above, we deleted DNA sequences essential for promoter activity within the LTR. SV40 virus stocks reconstructed with this mutated copy of the Harvey murine sarcoma virus LTR still transform mouse cells at an enhanced frequency. We speculate that when the LTR is placed more than 1.5 kilobases from the SV40 early promoter, the cis-acting enhancer element within the LTR can increase the ability of the SV40 promoter to effectively operate when integrated in a murine chromosome. These data are discussed in terms of the apparent cell specificity of viral enhancer elements.     

A recombinant Chinese hamster ovary cell line containing a 300-fold amplified tetramer of the hepatitis B genome together with a double selection marker expresses high levels of viral protein

A new series of double-selection plasmids containing recombinant genes expressing the neomycin phosphotransferase (NEO) of transposon Tn5 and mouse dihydrofolate reductase (DHFR) in mammalian cells is described. Activity of the recombinant DHFR gene varied more than 50-fold, depending on the location of the simian virus 40 72 base-pair repeat or enhancer, which is part of the promoter of the NEO unit. A NEO-DHFR module with the enhancer located at the 3' end of the DHFR gene was inserted into a plasmid containing four tandem head-to-tail copies of the hepatitis B virus (HBV) genome and the new plasmid was used to transform DHFR- Chinese hamster ovary cells. In one of the cell lines obtained, an unrearranged copy of the HBV tetramer could be amplified 300-fold by increasing selective pressure with methotrexate, resulting in a proportional increase of the synthesis of HBV surface antigen. Four different mRNAs detected in the amplified cell line probably encode HBV core protein, pre-S and surface antigens, and the X protein. As a result of the DNA amplification, synthesis of HBV proteins is no longer restricted to resting cells. Integrated plasmid sequences appear to be stable during the amplification process.     

Isolation of a functional human interleukin 2 gene from a cosmid library by recombination in vivo

A method has been developed that allows the isolation of genomic clones from a cosmid library by homologous recombination in vivo. This method was used to isolate a human genomic interleukin 2 (IL2) gene. The genomic cosmid library was packaged in vivo into lambda phage particles. A recombination-proficient host strain carrying IL2 cDNA sequences in a non-homologous plasmid vector was infected by the packaged cosmid library. After in vivo packaging and reinfection, recombinants carrying the antibiotic resistance genes of both vectors were selected. From a recombinant cosmid clone the chromosomal IL2 gene was restored. After DNA mediated gene transfer into mouse Ltk- cells human IL2 was expressed constitutively.     

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.     

Simplified cosmid vectors for gene transfer to cultured mammalian cells: isolation of the gene for elongation factor 2 from the mouse

We constructed a series of cosmid vectors that carry two tandemly arranged lambda cos and mammalian selective markers. We achieved cloning efficiencies of 1-3 x 10(7) and greater than 10(6) colony-forming units per microgram of insert, using a cloned 42-kb BamHI fragment and Sau3AI fragments of 40-50 kb from mouse genomic DNA, respectively. The modified Ca.phosphate coprecipitation method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607-616] considerably improved the efficiency of gene transfer of cosmids into cultured mammalian cells: when genes encoding thymidine kinase from herpes simplex virus type 1 and aminoglycoside 3'-phosphoribosyltransferase from Tn5 were selected, the efficiencies of gene transfer into mouse L cells were about 10(-6). The mouse genome contains one copy of the functional gene for elongation factor 2 (EF2) per haploid genome and multiple copies of the EF2-related gene. We isolated a cosmid that carried functional full-length mouse EF2 from a cosmid library of L-cell genomic DNA, by colony hybridization and subsequent gene transfer of candidate cosmids into human 143B cells.     

Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L cells

We have isolated a cDNA clone corresponding to a substantial portion of the human tissue-type plasminogen activator (t-PA) protein. It encodes almost all of the protein B chain and part of the 3' untranslated region. We have used this clone to screen bacteriophage lambda and cosmid libraries of human genomic DNA. Several related genomic clones were isolated. One of these, a cosmid clone, carried approx. 40 kb of human DNA. Mapping experiments indicate that the region containing the protein-coding exons is approx. 20 kb in length. The cosmid, containing the t-PA gene and the aminoglycosyl-3'-phosphotransferase dominant-selection marker, was introduced into mouse L cells. Approximately half of the transformants were shown to produce human t-PA. We demonstrated that the fibrinolytic t-PA activity could be specifically quenched by anti-t-PA antibody and that the recombinant t-PA was of similar size (by SDS-polyacrylamide gel electrophoresis) to the t-PA produced by the human Bowes melanoma cell line. Our results suggest that the cosmid clone carries the whole t-PA coding region together with the regulatory elements necessary for its expression.     

Cloning and expression of the chromosomal immune interferon gene of the rat.

The chromosomal immune interferon gene of the rat (IFN-gamma) was identified by screening a recombinant rat lambda phage library with a human IFN-gamma cDNA probe. In contrast to the genes of other rat IFNs, this rat IFN-gamma chromosomal gene contains introns and its structural organization closely resembles that of the human and murine IFN-gamma genes. The rat IFN-gamma gene encodes a signal sequence of 19 amino acids followed by the mature IFN-gamma protein of 137 amino acids. The gene was expressed under control of the simian virus 40 (SV40) early promoter in Chinese hamster ovary (CHO) cells deficient in dihydrofolate reductase (DHFR) after co-transformation with a plasmid containing the mouse DHFR gene. Initial transformants with a DHFR+ phenotype produced IFN-gamma titres ranging from 20 to 1600 units/ml. After stepwise increases in the concentration of methotrexate (MTX) in the growth medium of transformed CHO cells, MTX-resistant clones producing 80 000-100 000 units per ml were isolated. Protein analysis of supernatants of these MTX-resistant cells by polyacrylamide gel electrophoresis revealed a product with an apparent mol. wt. of 18 000 daltons which was not detectable in the growth medium of DHFR+ transformants that did not produce IFN. The product was identified as rat IFN-gamma and constituted approximately 5% of the proteins excreted from these cells.     

Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells.

Expression of human tissue-type plasminogen activator (t-PA) at high levels has been achieved in Chinese hamster ovary (CHO) cells by cotransfection and subsequent coamplification of the transfected sequences. Expression vectors containing the t-PA cDNA gene and dihydrofolate reductase (DHFR) cDNA gene were cotransfected into CHO DHFR-deficient cells. Transformants expressing DHFR were selected by growth in media lacking nucleosides and contained low numbers of t-PA genes and DHFR genes. Stepwise selection of the DHFR+ transformants in increasing concentrations of methotrexate generated cells which had amplified both DHFR genes and t-PA genes over 100-fold. These cell lines expressed elevated levels of enzymatically active t-PA. To optimize both t-PA sequence amplification and t-PA expression, various modifications of the original procedure were used. These included alterations to the DHFR expression vector, optimization of the molar ratio of t-PA to DHFR sequences in the cotransfection, and modification of the methotrexate resistance selection procedure. The structure of the amplified DNA, its chromosomal location, and its stability during growth in the absence of methotrexate are reported.     

Plasmid amplification-promoting sequences from the origin region of Chinese hamster dihydrofolate reductase gene do not promote position-independent chromosomal gene amplification

Initiation of DNA synthesis occurs with high frequency at oriß, a region of DNA from the amplified dihydrofolate reductase (DHFR) domain of Chinese hamster CHOC 400 cells that contains an origin of bidirectional DNA replication (OBR). Recently, sequences from DHFR oriß/OBR were shown to stimulate amplification of cis-linked plasmid DNA when transfected into murine cells. To test the role of oriß/OBR in chromosomal gene amplification, linearized plasmids containing these sequences linked to a DHFR expression cassette were introduced into DHFR- CHO DUKX cells. After selection for expression of DHFR, cell lines that contain a single integrated, unrearranged copy of the linearized expression plasmid were identified and exposed to low levels of the folate analog, methotrexate (MTX). Of seven clonal cell lines containing the vector control, three gained resistance to MTX by 5 to 15-fold amplification of the integrated marker gene. Of 16 clonal cell lines that contained oriß/OBR linked to a DHFR mini-gene, only 6 gained resistance to MTX by gene amplification. Hence, sequences from the DHFR origin region that stimulate plasmid DNA amplification do not promote amplification of an integrated marker gene in all chromosomal contexts. In addition to showing that chromosomal position has a strong influence on the frequency of gene amplification, these studies suggest that the mechanism that mediates the experiment of episomal plasmid DNA does not contribute to the early steps of chromosomal gene amplification.