Novel approaches to in vitro transgenesis

Abstract

The study of gene expression is a major focus in biological research and is recognised to be critical for our understanding of physiological and pathophysiological processes. Methods to study gene expression range from in vitro biochemical assays through cultured cells and tissue biopsies to whole organisms. In the early stages of project development, considerations about which model system to use should be addressed and may influence future experimental procedures. The aim of this review is to briefly describe advantages and disadvantages of the existing techniques available to study eukaryote gene expression in vitro, including the mechanism of transgene integration (transient or stable), the different transgenesis systems available, including plasmids, viruses and targeted integration and knockin approaches, and paying particular attention to expression systems such as bacterial artificial chromosomes and episomal vectors that offer a number of advantages and are increasing in popularity. We also discuss novel approaches that combine some of the above techniques, generating increasingly complex but physiologically accurate expression systems.

The Prolactin Gene: A Paradigm of Tissue‐Specific Gene Regulation with Complex Temporal Transcription Dynamics

“…New genomic technologies, as well as future developments, are also likely to affect experimental choices and may reduce animal use. For example, regulation of the human prolactin locus can now be studied using bacterial artificial chromosome (BAC) technologies, which has several advantages over the short DNA constructs used previously (14). These advantages include the ability to assess regulatory elements within their native context in combination with other regulatory elements within the locus, the ability to search for long‐range distal regulatory elements, the maintenance of chromatin influence on the locus, and the insulation of position effects from the DNA insertion site.…”

“…Emerging technologies will hopefully provide the resources needed to shed light on these unresolved issues. In particular, the increasing tractability of BAC transgenesis (14) will enable the use of larger constructs that will help to address how regulatory elements integrate their activity. Pulsatile expression patterns of the prolactin gene, probably alterable by tissue structure, also suggests the complex regulation of transcription activity and cell function.…”

Genetic Tools to Study Cardiovascular Biology

“…These were followed by second generation mouse lines using larger transgenes such as bacteriophage P1-derived Artificial Chromosomes (PACs, up to 120 Kb) and Bacterial Artificial Chromosomes (BACs, up to 250 Kb) that can carry significantly larger DNA sequences containing most if not all of a gene essential promoter/enhancer elements. These larger transgenes were also significantly less sensitive to genomic position and epigenetic variegation effects (Giraldo and Montoliu, 2001;Adamson et al, 2011). Regardless of their size, transgenes expression is less reliable when compared with direct knock-ins of a reporter or recombinase gene in the native locus of the cell type-specific gene.…”

A double-Flp-in method for stable overexpression of two genes

“…In drug research, cell lines overexpressing specific genes are an important screening tool. Overexpression can be achieved via numerous ways, with transient or stable expression of the gene of interest 3,4 . However, many transgene-introducing techniques come along with obvious disadvantages: Reproducibility, efficiency, and anisogeneity are only a few of them.…”