Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene

Kaufman, Randal J.; Sharp, Phillip A.

doi:10.1016/0022-2836(82)90103-6

Cited by 351 publications

(155 citation statements)

References 38 publications

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“…This relies on a gradual increase in the selection pressure over several months for a co-transfected selection gene product such as dihydrofolate reductase, and on mutant cells that require the expression of the selection gene for growth (Kaufman and Sharp, 1982;Schimke et al, 1982). More recent approaches to address the problem have included the isolation and integration in vectors of CHO cell sequences such as endogenous promoters and favorable integration site sequences, the identification of rare sites on the chromosome with high transcriptional activity combined with targeted transgene integration at one of these sites, the construction of artificial chromosomes, the improvement of the cell line selection and screening procedures, and the metabolic engineering of the recipient cells (Brezinsky et al, 2003;Csonka et al, 2000;Fussenegger et al, 1998Fussenegger et al, , 1999Koduri et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines

Girod

Zahn-Zabal

Mermod

2005

Biotechnol. Bioeng.

104

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Scaffold or matrix attachment region (S/MAR) genetic elements have previously been proposed to insulate transgenes from repressive effects linked to their site of integration within the host cell genome. We have evaluated their use in various stable transfection settings to increase the production of recombinant proteins such as monoclonal antibodies from Chinese hamster ovary (CHO) cell lines. Using the green fluorescent protein coding sequence, we show that S/MAR elements mediate a dual effect on the population of transfected cells. First, S/ MAR elements almost fully abolish the occurrence of cell clones that express little transgene that may result from transgene integration in an unfavorable chromosomal environment. Second, they increase the overall expression of the transgene over the whole range of expression levels, allowing the detection of cells with significantly higher levels of transgene expression. An optimal setting was identified as the addition of a S/MAR element both in cis (on the transgene expression vector) and in trans (co-transfected on a separate plasmid). When used to express immunoglobulins, the S/MAR element enabled cell clones with high and stable levels of expression to be isolated following the analysis of a few cell lines generated without transgene amplification procedures.

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Section: Introductionmentioning

confidence: 99%

Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines

Girod

Zahn-Zabal

Mermod

2005

Biotechnol. Bioeng.

104

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“…In both cell l 1 and Na2 genes, large amounts c RNA were also observed. These "read-through" transcription, abn activation of cryptic promoters, o ments, as previously demonstrate amplification studies (21,22 mRNA function in cell-free systems. Poly(A)+ RNA from the MTX-resistant cells was translated in vitro in either wheat germ extract or rabbit reticulocyte lysate, and the labeled products from these reactions were analyzed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis.…”

mentioning

confidence: 97%

“…In sta,ble transformants, the amplified DNA sequences usually reside in homogeneous staining regions of the chromosomes (11,34) some-like structures called double minutes (6,20). Subsequent studies showed that cloned DHFR genes and other genes cotransformed with them could also be amplified by this stepwise MTX selection when they were transferred into DHFR-deficient cells (10,21,22,32,35).…”

mentioning

confidence: 99%

Amplification and expression of human alpha-globin genes in Chinese hamster ovary cells.

Lau¹,

Lin²,

Kan³

1984

Mol. Cell. Biol.

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We studied the effects of gene amplification on human globin gene expression in Chinese hamster ovary cells. The normal human a-globin gene (Na2) and a hybrid gene (MaG) containing the 5' promoter-regulator region of the mouse metallothionein gene and the human structural aL2-globin gene were linked to a modular SV2-cDNA dihydrofolate reductase (DHFR) gene. The recombinant DNA molecules were introduced into Chinese hamster ovary cells by calcium phosphate precipitation. After initial selection to retain the DHFR and linked sequences, the cells were cultured in increasing concentrations of methotrexate up to 0.2 mM. Southern blot analysis of total cellular DNA showed an approximately 500-to 1,000-fold increase in the number of copies of DHFR and human ot-globin genes. The transcription of the a-globin and DHFR genes increased as their copy number within the cells increased. The transcription of the amplified hybrid MaG gene was also inducible with cadmium treatments. Both mature mRNA and "read-through" transcripts were observed. DHFR constituted approximately 10% of pulse-labeled cellular proteins in these cells, but no human a-globin was detected. In vitro translation of polyadenylated RNA from these cells showed that a-globin mRNA transcribed from the amplified a-globin genes was functional and directed a-globin chain synthesis. In situ hybridization of 3H-labeled oa-globin and DHFR DNA probes in chromosome preparations from the two cell lines indicated that both genes were coamplified in the same chromosomal locations in each cell type. These results indicate that gene amplification enhances human globin gene expression in cultured Chinese hamster ovary cells.

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“…To generate a more homogenous form of Dlp (Dlp ΔNCF ), the gene encoding a Dlp fragment spanning residues 74-617 but lacking residues 400-437, which intervene between the two sites of partial proteolytic processing, and with Asn 79 and Asn 502 substituted with glutamate to remove the two consensus N-linked glycosylation attachments, was subcloned into pSGHV0 and stably tranfected into dhfr −∕− CHO cells. Dlp ΔNCF expression levels were amplified by selection of cell lines in methotrexate (46), and Dlp ΔNCF purified from conditioned medium using immobilized metal ion affinity, anion exchange, and size-exclusion chromatographies. Final yields of purified Dlp ΔNCF were 1-2 mg per liter of conditioned medium.…”

Section: Methodsmentioning

confidence: 99%

Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling

Kim

Saunders

Hamaoka

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Glypicans are heparan sulfate proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core region followed by a stalk region that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk regions are predicted to be random coil but contain a variable number of attachment sites for heparan sulfate chains. Both the N-terminal protein core and the heparan sulfate attachments are important for glypican function. We report here the 2.4-Å crystal structure of the N-terminal protein core region of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, α-helical fold for glypican core regions that does not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and we identify a localized region on the Dlp surface important for mediating its function in Hh signaling. Purified Dlp protein core does not, however, interact appreciably with either Hh or an Hh:Ihog complex.

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Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene

Cited by 351 publications

References 38 publications

Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines

Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines

Amplification and expression of human alpha-globin genes in Chinese hamster ovary cells.

Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling

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