Transfer and Expression of Foreign Genes in Mammalian Cells

Abstract

The transfer of foreign genes into eukaryotic cells, in particular mammalian cells, has been essential to our understanding of the functional significance of genes and regulatory sequences as well as the development of gene therapy strategies. To this end, different mammalian expression vector systems have been designed. The choice of a particular expression system depends on the nature and purpose of the study and will involve selecting particular parameters of expression systems such as the type of promoter/enhancer sequences, the type of expression (transient versus stable) and the level of desired expression. In addition, the success of the study depends on efficient gene transfer. The purification of the expression vectors, as well as the transfer method, affects transfection efficiency. Numerous approaches have been developed to facilitate the transfer of genes into cells via physical, chemical or viral strategies. While these systems have all been effective in vitro they need to be optimized for individual cell types and, in particular, for in vivo transfection.

Expression of ΔF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR

“…16,26 Given the observation that at least one mouse per experiment showed successful modification with lipid vehicles, in vitro experiments can be useful for defining some of the conditions for effective in vivo DNA transfer with lipids. The ultimate optimization of transfection efficiency will; however, require analysis in vivo.…”

“…For the experiments presented here, the transfer conditions were established as follows. Given the already overwhelming body of literature on different compounds for the delivery of DNA in vivo, 16,17 the delivery vehicles that were chosen have either been previously used in vitro or have been tested for in vivo delivery into the airways with reporter genes. The decision for target choice was based on the fact that the primary cause of death and morbidity in CF is due to lung infections; therefore, the development of an SFHR-based gene therapy for CF would include delivery into the lung.…”

Illegitimate DNA integration in mammalian cells

“…Many transfection procedures have been developed that enable DNA to enter into the cytoplasm, but its passage into the nucleus is mainly mediated by cellular processes. 1,2 After its entry into the nucleus, a large proportion of the foreign DNA is rapidly degraded or diluted among subsequent cell divisions. 3 However, molecules containing an origin of replication (typically derived from viruses) can persist for long periods as extrachromosomally replicating episomes under certain conditions (such as expression of viral tumour antigens or association with the nuclear matrix).…”

The SET domain protein Metnase mediates foreign DNA integration and links integration to nonhomologous end-joining repair

“…It is occasionally used advantageously in gene therapy of human disease, such as in retroviral correction of inherited immunologic deficiencies (3,4). Of much wider utility, it has been a foundation of molecular laboratory investigation for decades, allowing for stable modification of a cell's genomic content for construction of in vivo and in vitro laboratory models (5,6).…”