AAV-mediated gene targeting.
The precise alteration of sequences by homologous recombination is an important strategy for gene therapies as well as investigating gene function and cellular DNA repair pathways. Inefficient delivery of template DNA to the nucleus using transfection or electroporation methods is one limitation of the frequency of homologous recombination in primary cells. AAV vectors can be used to efficiently deliver single stranded DNA recombination templates to the nucleus of primary cells and the AAV genome structure with single DNA strands stabilized by inverted terminal repeat sequences is likely one reason for the increase in recombination frequencies observed. Thus, an AAV-mediated gene targeting approach allows cells from normal or disease-affected individuals to be modified for careful study. When clones of primary cells can be expanded, autologous transplantation of phenotypically corrected cells is also feasible. Here we describe a basic approach to gene targeting using an AAV-mediated strategy. Vector design strategies are discussed, and protocols for altering expressed and non-expressed loci in primary somatic cells, and stem cells are reviewed.
Department of Pediatrics, University of Washington, Seattle, WA, USA. dgmiller@u.washington.edu
Daniel G
DG
Miller
D015139:Blotting, Southern; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans