Methodology article

Gene targeting in adult rhesus macaque fibroblasts

Daniel T Meehan^{* 1} , Mary Ann Zink^{* 1} , Melissa Mahlen¹ , Marilu Nelson² , Warren G Sanger² , Shoukhrat M Mitalipov³ , Don P Wolf³ , Michel M Ouellette⁴ and Robert B Norgren Jr¹

¹Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, 985805 Nebraska Medical Center, Omaha, NE 68198-5805, USA

²Department of Pediatrics and Human Genetics Laboratory, Munroe Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center, Omaha 68198-5440, USA

³Oregon National Primate Research Center, Oregon Stem Cell Center, Oregon Health & Science University, 505 NW 185th Ave, Beaverton, OR 97006, USA

⁴Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, NE 68198, USA

author email corresponding author email* Contributed equally

BMC Biotechnology 2008, 8:31doi:10.1186/1472-6750-8-31

Published:	26 March 2008

Abstract

Background

Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases. Successful gene targeting in fibroblasts followed by somatic cell nuclear transfer (SCNT) has been achieved in several species of large mammals but not yet in primates. Our goal was to establish the protocols necessary to achieve gene targeting in primary culture of adult rhesus macaque fibroblasts as a first step in creating nonhuman primate models of genetic disease using nuclear transfer technology.

Results

A primary culture of adult male fibroblasts was transfected with hTERT to overcome senescence and allow long term in vitro manipulations. Successful gene targeting of the HPRT locus in rhesus macaques was achieved by electroporating S-phase synchronized cells with a construct containing a SV40 enhancer.

Conclusion

The cell lines reported here could be used for the production of null mutant rhesus macaque models of human genetic disease using SCNT technology. In addition, given the close evolutionary relationship and biological similarity between rhesus macaques and humans, the protocols described here may prove useful in the genetic engineering of human somatic cells.