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. 1999 Jan 5;96(1):127-32.
doi: 10.1073/pnas.96.1.127.

Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector

J P Condreay  1 , S M Witherspoon, W C Clay, T A Kost
Affiliations

Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector

J P Condreay et al. Proc Natl Acad Sci U S A. .

Abstract

Recombinant baculoviruses can serve as gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types. Furthermore, by inclusion of a dominant selectable marker in the viral vector, cell lines can be derived that stably express recombinant genes. A virus was constructed containing two expression cassettes controlled by constitutive mammalian promoters: the cytomegalovirus immediate early promoter/enhancer directing expression of green fluorescent protein and the simian virus 40 (SV40) early promoter controlling neomycin phosphotransferase II. Using this virus, efficient gene delivery and expression was observed and measured in numerous cell types of human, primate, and rodent origin. In addition to commonly used transformed cell lines such as HeLa, CHO, Cos-7, and 293, this list includes primary human keratinocytes and bone marrow fibroblasts. In all cases, addition of butyrate or trichostatin A (a selective histone deacetylase inhibitor) to transduced cells markedly enhanced the levels of reporter protein expression observed. When transduced cells are put under selection with the antibiotic G418, cell lines can be obtained at high frequency that stably maintain the expression cassettes of the vector DNA and exhibit stable, high-level expression of the reporter gene. Stably transduced derivatives have been selected from a substantial number of different cell types, suggesting that stable lines can be derived from any cell type that exhibits transient expression.

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Figures

Figure 1
Figure 1
Shuttle vector used to construct BacMam1 GFP baculovirus. A detailed description is in the text.
Figure 2
Figure 2
Transduction of CHO cells with BacMam1 GFP baculovirus. (A and B) Visualization of GFP-expressing cells by fluorescence microscopy. (A) Cells transduced with BacMam1 GFP. (B) Same as A except for addition of 1 mM butyrate after incubation with virus. (C) Analysis of GFP expression by flow cytometry. Black line, nontransduced cells; blue line, transduced cells; red line, transduced cells plus 1 mM butyrate; green line, same as red line but with neutral-density filter in place.
Figure 3
Figure 3
Western blot analysis of GFP and NPT II expression in CHO cells. Extracts of cells were prepared 24 hr posttransduction. Lanes 1–3, cells probed with antibody directed against GFP; lanes 4–6, probed with antibody directed against NPT II. Lanes 1 and 4, nontransduced cells; lanes 2 and 5, transduced cells; lanes 3 and 6, transduced cells plus 1 mM butyrate. KDa, kilodaltons.
Figure 4
Figure 4
Detection of baculovirus particles in CHO cells by electron microscopy. (A) Virus particle at the surface of cell. (×85,000.) (B) Virus particles enclosed in membrane vesicle. (×73,000.) Arrows denote virus particles. PM, plasma membrane; N, nucleus.
Figure 5
Figure 5
Effect of TSA on GFP expression in CHO cells transduced with BacMam1 GFP baculovirus. Transduced cells were incubated for 24 hr in the presence of the indicated concentrations of TSA and GFP quantified by using flow cytometry. Results are the average of three independent transductions.
Figure 6
Figure 6
Formation of stably transduced CHO cells with BacMam1 GFP. (A–C) Effect of virus inoculum on number of G418-resistant clones. Cells were transduced with virus at different moi and clones were selected as described in the text. (A) moi of 1. (B) moi of 10. (C) moi of 100. (D) Fluorescence micrograph of a stably transduced clone.
Figure 7
Figure 7
Analysis of baculovirus-derived DNA in stably transduced CHO cells. (A) Map of BacMam1 GFP recombinant baculovirus DNA. StuI recognition sites internal and adjacent to the recombinant expression cassettes are shown with sizes of the expected fragments. Drawing is not to scale. (B) Southern blot of StuI-digested DNA. Details are described in the text. Lanes: 1, nontransduced cells; 2, transduced cells, selected pool; 3, BacMam1 GFP viral DNA.
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References

    1. Volkman L E, Goldsmith P A. Appl Environ Microbiol. 1983;45:1085–1093. - PMC - PubMed
    1. Carbonell L F, Miller L K. Appl Environ Microbiol. 1987;53:1412–1417. - PMC - PubMed
    1. Hofmann C, Sandig V, Jennings G, Rudolph M, Schlag P, Strauss M. Proc Natl Acad Sci USA. 1995;92:10099–10103. - PMC - PubMed
    1. Boyce F M, Bucher N L. Proc Natl Acad Sci USA. 1996;93:2348–2352. - PMC - PubMed
    1. Shoji I, Aizaki H, Tani H, Ishii K, Chiba T, Saito I, Miyamura T, Matsuura Y. J Gen Virol. 1997;78:2657–2664. - PubMed

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