An efficient system for characterizing the expression of cloned mammalian genes is DNA-mediated gene transfer into cultured heterologous cells. This chapter outlines the protocols for cloning and expression of the human apolipoprotein E (apoE) gene isolated from available genomic libraries that have been constructed in both λ and cosmid vectors. Cosmid vectors permit the direct use of an isolated clone in gene transfection without additional manipulation. Human apoE gene-containing cosmid DNA is introduced into mouse L cells by a modification of the calcium phosphate coprecipitation method. The chapter indicates that the level of human apoE mRNA expressed by the exogenous gene in different stable cell lines has been found to vary greatly, from undetectable to amounts comparable to that observed in the human liver. Thus the availability of the cloned human apoE gene and knowledge of its structure will facilitate a direct examination of the molecular basis of its regulation, and expression of the isolated gene in transfected cells will permit the biological assay of specific reconstructions designed to correlate particular sequences with their functions.
All Science Journal Classification (ASJC) codes
- Molecular Biology