Targeted Transgenesis

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 17; pp. 8804 - 8808
• Main Authors: Jasin, Maria, Moynahan, Mary Ellen, Richardson, Christine
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 20.08.1996; National Academy of Sciences
• Subjects: Animals; Commentary; DNA; Gene Targeting - methods; Gene transfer techniques; Genes; Genetic loci; Genomes; Germ cells; Hypoxanthine Phosphoribosyltransferase - genetics; Mice; Pluripotent stem cells; Promoter Regions, Genetic; Stem cells; Transgenes; Transgenic animals; X Chromosome - genetics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.17.8804

The decade of the 1980s witnessed a revolution in mouse molecular genetics. At its start, the first transgenic mouse was created by nuclear injection of cloned DNA into fertilized mouse eggs. This built on earlier studies in which viruses carried DNA into mice. With nuclear injection of DNA, investigators rapidly reported germ-line transmission of transgenes, expression of transgenes, and a phenotype associated with transgene expression. Subsequently, in a flurry of studies that have continued unabated, transgenic mice have been used to study a number of phenomena, including tissue-specific gene expression, oncogenesis, and developmental mutations. Since injected DNA primarily integrates at random locations in the genome, transgene insertion by microinjection is usually restricted to gain-of-function studies in the mouse. By the end of the decade, the second major advance in mouse molecular genetics had occurred-the disruption of genes in mice. Key to this approach was the ability to grow pluripotential embryonic stem (ES) cells in culture. If their pluripotency is maintained, ES cells can be returned to mouse embryos and contribute to all tissues of a mouse, including the germ line. ES cells were modified to carry foreign DNA into mice using standard gene transfer approaches and could also be manipulated to carry a selectable mutation into mice. Most significant, however, was the application of gene targeting to these cells, resulting in the creation of loss-of-function mutations in the mouse. With gene targeting, the homologous integration of DNA into the genome, any cloned sequence can be altered in the genome. Several hundred "knock-out" mice have now been created. These two approaches, gain-of-function transgenesis and gene targeting in ES cells, have now been combined. The report by Bronson et al. describes the introduction of transgenes by gene targeting into the hypoxanthine phosphoribosyltransferase (hprt) locus of ES cells. The transgenes consist of a mouse bcl-2 cDNA driven by either the chicken or human beta -actin promoter. Gene targeting of transgenes controlled both the site and copy number of the transgene insertion. The results presented in this report are convincing that targeted integrations of a transgene, in contrast to random integrations, yield consistent expression levels of transgenes both in cell clones and in mouse tissues. Targeting transgenes to loci in ES cells, therefore, may be a much improved method for controlling their expression.