scholarly journals A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells.

1983 ◽  
Vol 3 (2) ◽  
pp. 280-289 ◽  
Author(s):  
H Okayama ◽  
P Berg
Keyword(s):  
Cdna Cloning ◽  
Mammalian Cells ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Plasmid Vector ◽  
Simian Virus ◽  
Cdna Clones ◽  
Alpha Globin ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Cloned Cdna

This paper describes a plasmid vector for cloning cDNAs in Escherichia coli; the same vector also promotes expression of the cDNA segment in mammalian cells. Simian virus 40 (SV40)-derived DNA segments are arrayed in the pcD vector to permit transcription, splicing, and polyadenylation of the cloned cDNA segment. A DNA fragment containing both the SV40 early region promoter and two introns normally used to splice the virus 16S and 19S late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts. An SV40 late region polyadenylation sequence occurs downstream of the cDNA cloning site, so that the cDNA transcript acquires a polyadenylated 3' end. By using pcD-alpha-globin cDNA as a model, we confirmed that the alpha-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the cDNA segment or at the distal SV40 polyadenylation signal. A cDNA clone library constructed with mRNA from SV40-transformed human fibroblasts and this vector (about 1.4 X 10(6) clones) yielded full-length cDNA clones that express hypoxanthine-guanine phosphoribosyltransferase (Jolly et al., Proc. Natl. Acad. Sci. U.S.A., in press).

A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells

1983 ◽  
Vol 3 (2) ◽  
pp. 280-289
Author(s):  
H Okayama ◽  
P Berg
Keyword(s):  
Cdna Cloning ◽  
Mammalian Cells ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Plasmid Vector ◽  
Simian Virus ◽  
Cdna Clones ◽  
Alpha Globin ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Cloned Cdna

This paper describes a plasmid vector for cloning cDNAs in Escherichia coli; the same vector also promotes expression of the cDNA segment in mammalian cells. Simian virus 40 (SV40)-derived DNA segments are arrayed in the pcD vector to permit transcription, splicing, and polyadenylation of the cloned cDNA segment. A DNA fragment containing both the SV40 early region promoter and two introns normally used to splice the virus 16S and 19S late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts. An SV40 late region polyadenylation sequence occurs downstream of the cDNA cloning site, so that the cDNA transcript acquires a polyadenylated 3' end. By using pcD-alpha-globin cDNA as a model, we confirmed that the alpha-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the cDNA segment or at the distal SV40 polyadenylation signal. A cDNA clone library constructed with mRNA from SV40-transformed human fibroblasts and this vector (about 1.4 X 10(6) clones) yielded full-length cDNA clones that express hypoxanthine-guanine phosphoribosyltransferase (Jolly et al., Proc. Natl. Acad. Sci. U.S.A., in press).

scholarly journals High-efficiency transformation of mammalian cells by plasmid DNA.

1987 ◽  
Vol 7 (8) ◽  
pp. 2745-2752 ◽  
Author(s):  
C Chen ◽  
H Okayama
Keyword(s):  
Calcium Phosphate ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Efficient Transformation ◽  
Transfection Protocol ◽  
Hela Cell Lines ◽  
Level 3 ◽  
Cloned Cdna

We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.

High-efficiency transformation of mammalian cells by plasmid DNA

1987 ◽  
Vol 7 (8) ◽  
pp. 2745-2752
Author(s):  
C Chen ◽  
H Okayama
Keyword(s):  
Calcium Phosphate ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Efficient Transformation ◽  
Transfection Protocol ◽  
Hela Cell Lines ◽  
Level 3 ◽  
Cloned Cdna

We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.

Comparison of intron-dependent and intron-independent gene expression

1988 ◽  
Vol 8 (10) ◽  
pp. 4395-4405
Author(s):  
A R Buchman ◽  
P Berg
Keyword(s):  
Simian Virus 40 ◽  
Plasmid Vector ◽  
Fold Increase ◽  
Simian Virus ◽  
Transcription Unit ◽  
Vector System ◽  
Beta Globin ◽  
Animal Cells ◽  
Transcriptional Enhancer ◽  
Kinase Gene

Recombinant simian virus 40 viruses carrying rabbit beta-globin cDNA failed to express the beta-globin sequence unless an intron was included in the transcription unit. The addition of either beta-globin IVS1 or IVS2 caused a 400-fold increase in RNA production. Stable beta-globin RNA production required sequences in IVS2 that were very close to the splice sites and that coincided with those needed for mRNA splicing. In addition to the recombinant viruses, intron-dependent expression was observed with both replicating and nonreplicating plasmid vectors in short-term transfections of cultured animal cells. Unlike transcriptional enhancer elements, IVS2 failed to increase stable RNA production when it was placed downstream of the polyadenylation site. Using a plasmid vector system to survey different inserted sequences for their dependence on introns for expression, we found that the presence of IVS2 stimulated the expression of these sequences 2- to 500-fold. Sequences from the transcribed region of the herpes simplex virus thymidine kinase gene, a gene that lacks an intervening sequence, permitted substantial intron-independent expression (greater than 100-fold increase) in the plasmid vector system.

Stimulation of the acute-phase response in simian virus 40-hepatocyte cell lines

1989 ◽  
Vol 9 (7) ◽  
pp. 2779-2786
Author(s):  
W S Liao ◽  
K T Ma ◽  
C D Woodworth ◽  
L Mengel ◽  
H C Isom
Keyword(s):  
Cell Lines ◽  
Acute Phase ◽  
Molecular Mechanisms ◽  
Constitutive Expression ◽  
Simian Virus 40 ◽  
Normal Liver ◽  
Simian Virus ◽  
Cdna Clones ◽  
Dependent Manner ◽  
Amyloid A

Seven simian virus 40 (SV40)-hepatocyte cell lines were characterized with respect to the ability to express eight liver acute-phase genes. cDNA clones corresponding to albumin, serum amyloid A, alpha 1-acid glycoprotein, haptoglobin, alpha-, beta-, and gamma-fibrinogen, and alpha 1-major-acute-phase protein mRNAs were used in Northern (RNA) or slot blot analyses. In the noninduced state, six of the seven cell lines showed significant (i.e., liverlike) levels of constitutive expression of all genes examined except that expression of haptoglobin mRNA was considerable lower than in the normal liver. To examine whether these immortalized liver cells can respond appropriately to inflammatory mediators, cells were treated with conditioned medium from activated human monocytes or mixed lymphocyte cultures. Results showed that these SV40-hepatocyte cell lines responded to the conditioned media in culture by down-regulating albumin gene expression and up-regulating other acute-phase genes in a time- and dose-dependent manner. These results indicate that the SV40-hepatocytes retained not only the ability to express a number of acute-phase genes but also the ability to respond to external stimuli. The usefulness of these cell lines for analysis of the molecular mechanisms involved in the regulation of these acute-phase genes is discussed.

Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction

1986 ◽  
Vol 6 (12) ◽  
pp. 4295-4304
Author(s):  
D B Roth ◽  
J H Wilson
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Restriction Enzymes ◽  
Simian Virus ◽  
T Antigen ◽  
Short Sequence ◽  
End Joining ◽  
Nonhomologous Recombination ◽  
Sequence Homologies ◽  
Linear Molecules

Although DNA breakage and reunion in nonhomologous recombination are poorly understood, previous work suggests that short sequence homologies may play a role in the end-joining step in mammalian cells. To study the mechanism of end joining in more detail, we inserted a polylinker into the simian virus 40 T-antigen intron, cleaved the polylinker with different pairs of restriction enzymes, and transfected the resulting linear molecules into monkey cells. Analysis of 199 independent junctional sequences from seven constructs with different mismatched ends indicates that single-stranded extensions are relatively stable in monkey cells and that the terminal few nucleotides are critical for cell-mediated end joining. Furthermore, these studies define three mechanisms for end joining: single-strand, template-directed, and postrepair ligations. The latter two mechanisms depend on homologous pairing of one to six complementary bases to position the junction. All three mechanisms operate with similar overall efficiencies. The relevance of this work to targeted integration in mammalian cells is discussed.

Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants

1986 ◽  
Vol 6 (7) ◽  
pp. 2704-2711 ◽  
Author(s):  
D S Peabody ◽  
S Subramani ◽  
P Berg
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Initiation Codon ◽  
Efficient Production ◽  
Translation Efficiency ◽  
Reading Frame ◽  
Recombinant Viruses ◽  
Internal Translation ◽  
Late Region

In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation.

Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells

1985 ◽  
Vol 5 (8) ◽  
pp. 2080-2089
Author(s):  
C T Wake ◽  
F Vernaleone ◽  
J H Wilson
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Animal Cells ◽  
Linear Molecules ◽  
Sv40 Genome ◽  
Foreign Dna ◽  
The Individual ◽  
Sv40 Dna

Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed.

scholarly journals Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells.

1985 ◽  
Vol 5 (8) ◽  
pp. 2080-2089 ◽  
Author(s):  
C T Wake ◽  
F Vernaleone ◽  
J H Wilson
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Animal Cells ◽  
Linear Molecules ◽  
Sv40 Genome ◽  
Foreign Dna ◽  
The Individual ◽  
Sv40 Dna

Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed.

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