Gene targeting in adult rhesus macaque fibroblasts

Robert B Norgren,Daniel T Meehan,Melissa Mahlen,Warren G Sanger,Mary Ann Zink,Shoukhrat M Mitalipov,Marilu Nelson,Michel M Ouellette,Don P Wolf

doi:10.1186/1472-6750-8-31

Abstract

BackgroundGene 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.ResultsA 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.ConclusionThe 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.

Highlights

Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases
To extend the time that fibroblasts could be cultured, we electroporated adult male rhesus macaque fibroblasts with a construct containing a human telomerase reverse transcriptase expression cassette
To determine if human telomerase reverse transcriptase (hTERT) was expressed in the rhesus fibroblasts, RNA was extracted from neomycin resistance (NEOr) cells and reverse transcribed to cDNA

Summary

Introduction

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. The vast majority of genetic models of human disease in mammals have utilized the mouse as an experimental animal [7]. This is due to the relative ease with which genetically modified animals, including loss-offunction and knock-in mutations, can be produced by transfecting ES cells and creating chimeric animals with germline changes [7]. ES cells have been derived from primates including rhesus macaques, they have not been shown to contribute to chimeras

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