In theory, genetic manipulation is the only permanent method of altering the biochemistry of somatic cells. In practice, the concept faces many hurdles, not least being a safe and accurate method of delivering the right gene to the right place. In mice, for example, the use of Cre recombinase to splice out specific genes flanked by loxP DNA sequences, has been used with some success. However, this methodology requires crossing the Cre-expressing mice with those harboring loxP sites. Now, a paper in the February 12 PNAS online, demonstrates not only spatial, but temporal accuracy, in delivering Cre to, and ablating gene targets in, mouse somatic cells.
The technique, developed at Fred Gage's lab at the Salk Institute, La Jolla, California, combines the power for gene recombination of the Cre/loxP system with the low toxicity of the adeno-associated virus (AAV). The authors constructed an AAV vector containing a green fluorescent protein (GFP)-Cre fusion gene, driven by the cytomegalovirus (CMV) promoter. They first tested the vector in vitro, using HEK293 cells transfected with an inactive b-gal reporter gene; this gene is interrupted by a loxP-flanked stuffer region, which causes early termination of β-gal transcription and can be ablated by Cre-driven recombination. Expression of Cre in the cells was confirmed by GFP fluorescence, and recombination by expression of β-galactosidase. They then used the AAV in ROSA26 reporter mice, which have a similarly interrupted LacZ gene.—Tom Fagan
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- Kaspar BK, Vissel B, Bengoechea T, Crone S, Randolph-Moore L, Muller R, Brandon EP, Schaffer D, Verma IM, Lee KF, Heinemann SF, Gage FH. Adeno-associated virus effectively mediates conditional gene modification in the brain. Proc Natl Acad Sci U S A. 2002 Feb 19;99(4):2320-5. PubMed.