Experimental manipulation of gene expression / edited by Masayori Inouye.

Date:
1983
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Experimental manipulation of gene expression / edited by Masayori Inouye. Source: Wellcome Collection.

    174/344 (page 156)
    156 R. С. Mulligan 1. Transient expression assays (Banerji et al., 1981; Mellon et al., 1981), in which gene expression is assessed shortly after introducing the cloned sequences into cells (usually 12-72 hr after transfection or microinjec¬ tion) 2. Stable transformation (Wigler a/., 1977; Mulligan and Berg, 1981a), in which the DNA sequence of interest is stably introduced into the recipient cell, most often through integration of the exogenous sequence into chro¬ mosomal DNA of the recipient. These systems have already become extremely valuable in the definition of common nucleotide sequences required for the expression of most, if not all, eukaryotic genes (McKnight et al., 1981; Benoist and Chambón, 1981; Fromm and Berg, 1983), and they are beginning to shed light on the nature of DNA sequences involved in the regulated expression of specific genes (Huang et al., 1981; Lee et al., 1981; Mellon et al., 1983). Unfortunately, a major obstacle to this latter work may prove to be the ex¬ tremely variable transformation frequencies obtained with different cells. Al¬ though it appears that many common cell lines used in the laboratory can be readily transformed using a number of standard transformation protocols (Graham and Van der Eb, 1973; Parker and Stark, 1979) and available selectable markers and selective schemes (Wigler et al., 1977; Mulligan and Berg, 1981a; Southern and Berg, 1982), transformation frequencies for a number of spe¬ cialized cell lines and primary tissues can be dramatically lower (some cells appear totally refractory to DNA-mediated transformation). This variable effi¬ ciency has severely restricted the range of recipients available for experiments that require a specific host cell in order to demonstrate the regulated expression of a cloned gene (Mellon et al., 1983). Furthermore, it bodes ill both for the prospect of introducing sequences into a minor component of a heterogeneous cell population and for introducing whole populations of cloned sequences into cells. In an attempt to overcome these limitations, we set out to develop a more powerful system for gene transfer in mammalian cells. We chose to begin genetic manipulations to convert the murine retroviruses to highly transmissible agents for the transfer of exogenous genes, principally because a number of features of the retrovirus life cycle (Weiss, 1982) appeared uniquely suited for gene transfer both in vitro and in vivo: 1. Although the viral genome is RNA, during the viral life cycle a DNA- intermediate is formed that is extremely efficiently integrated into the chromosomal DNA of the infected cells (in an invariant fashion with respect to the viral genome). 2. Mammalian cells are not generally killed by productive infection by retroviruses.