Published online 11 February 2004
Nucleic Acids Research, 2004, Vol. 32, No. 3 e25
© 2004 Oxford University Press
Nuclear localization signal and cell synchrony enhance gene targeting efficiency in primary fetal fibroblasts
Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, 620 Hutton Street, Raleigh, NC 27606, USA
*To whom correspondence should be addressed. Tel: +1 919 515 7407; Fax: +1 919 515 4237; Email: Jorge_Piedrahita{at}ncsu.edu
The use of primary somatic cells in nuclear transfer procedure has opened a new opportunity to manipulate domestic animal genomes via homologous recombination. To date, while a few loci have been targeted in somatic cells using similar enrichment strategies as those used in mouse ES cells, there have been problems of low efficiency, mixed targeted and non-targeted cells within a colony and difficulties in cloning the cell after targeting. Utilizing the hypoxanthine guanine phosphoribosyl transferase (HPRT) as a test locus, it was determined that while no targeted colonies were identified using a conventional targeting construct, an average of 1 per million targeted cells were identified when a nuclear localization signal (nls) was added to the construct. When the nls was combined with cell synchronization using a thymidine block, targeting efficiency increased 7-fold. Moreover, the number of random integrants decreased by over 54-fold resulting in a 1:3 targeted to random integration ratio. This method should facilitate the application of homologous recombination to primary somatic cells.
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