Cloned Genes Transferred into Cultured Cells Allow in Vitro Analysis of Gene Function

Diverse techniques have been developed for transfecting genes into cells. A common technique involves the use of a retrovirus in which a viral structural gene has been replaced with the cloned gene to be transfected. The altered retrovirus is then

Transfect into cells mRNA CAT enzyme

Lyse cells

Lyse cells

Add [14C] chloramphenicol and acetyl-CoA

Incubate at 37°

Inactive Active promoter promoter

Autoradiogram of thin-layer chromatogram

FIGURE 23-13

Inactive Active promoter promoter

Autoradiogram of thin-layer chromatogram

Acetylated chloramphenicol

Chloramphenicol

CAT assay for assessing functional activity of a promoter sequence. In this assay, a DNA construct consisting of the promoter of interest and the reporter gene encoding chloramphenicol acetyltransferase (CAT) is introduced (transfected) into eukaryotic cells. If the promoter is active, the CAT gene will be transcribed and the CAT enzyme will be produced within the transfected cell. The presence of the enzyme can easily be detected by lysing the cell and incubating the cell lysate with [14C] chloramphenicol and acetyl-CoA. If present, the CAT enzyme will transfer the acetyl group from acetyl-CoA to the chloramphenicol, forming acetylated chloramphenicol, which can be easily detected by thin-layer chromatography. [Adapted from J. D. Watson et al, 1992, Recombinant DNA, 2nd ed, W. H. Freeman and Company.]

Acetylated chloramphenicol

Chloramphenicol

CAT assay for assessing functional activity of a promoter sequence. In this assay, a DNA construct consisting of the promoter of interest and the reporter gene encoding chloramphenicol acetyltransferase (CAT) is introduced (transfected) into eukaryotic cells. If the promoter is active, the CAT gene will be transcribed and the CAT enzyme will be produced within the transfected cell. The presence of the enzyme can easily be detected by lysing the cell and incubating the cell lysate with [14C] chloramphenicol and acetyl-CoA. If present, the CAT enzyme will transfer the acetyl group from acetyl-CoA to the chloramphenicol, forming acetylated chloramphenicol, which can be easily detected by thin-layer chromatography. [Adapted from J. D. Watson et al, 1992, Recombinant DNA, 2nd ed, W. H. Freeman and Company.]

used as a vector for introducing the cloned gene into cultured cells. Because of the properties of retroviruses, the recombinant DNA integrates into the cellular genome with a high frequency. In an alternative method, the cloned gene of interest is complexed with calcium phosphate. The calcium-phosphate-DNA complex is slowly precipitated onto the cells and the DNA is taken up by a small percentage of them. In another transfection method, called electroporation, an electric current creates pores in cell membranes through which the cloned DNA is taken up. In both of these latter methods, the trans-

Class II MHC a chain DNA

Class II MHC a chain DNA

Fibroblasts

Transfection

Fibroblasts

Transfection

Selection with G418 and mycophenolic acid

Selection with G418 and mycophenolic acid

Class II MHC

fected DNA integrates, apparently at random sites, into the DNA of a small percentage of treated cells.

Generally, the cloned DNA being transfected is engineered to contain a selectable marker gene, such as one that confers resistance to neomycin. After transfection, the cells are cultured in the presence of neomycin. Because only the transfected cells are able to grow, the small number of transfected cells in the total cell population can be identified and selected.

Transfection of cloned genes into cells has proved to be highly effective in immunologic research. By transfecting genes involved with the immune response into cells that lack those genes, the product of a specific gene can be studied apart from interacting proteins encoded by other genes. For example, transfection of MHC genes, under the control of appropriate promoters, into a mouse fibroblast cell line (L929, or simply L, cells) has enabled immunologists to study the role of MHC molecules in antigen presentation to T cells (Figure 23-14). Transfection of the gene that encodes the T-cell receptor has

FIGURE 23-14

Transfection of the genes encoding the class II MHC a chain and p chain into mouse fibroblast L cells, which do not normally produce these proteins. Two constructs containing one of the MHC genes and a selectable gene were engineered: the a-chain gene with the guanine phosphoribosyl transferase gene (gpt), which confers resistance to the drug G418, and the p-chain gene with a neomycin gene (neo), which confers resistance to mycophenolic acid. After transfection, the cells are placed in medium containing both G418 and mycophenolic acid. Only those fibroblasts containing both the neo and gpt genes (and consequently the genes encoding the class II MHC a and P chains) will survive this selection. These fibroblasts will express both class II MHC chains on their membranes.

provided information about the antigen-MHC specificity of the T-cell receptor.

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