Targeted Modification Of CCR5Gene In Rabbits And Albumin Gene In Pigs By TALENs

Transcription activator-like effector nuclease (TALEN) is a newly developedtechnology that can be used for genome-targeted modifications. The design andconstruction of TALENs is based on transcription activator-like effector (TALE),which was originally discovered in the plant pathogen Xanthomonas. TALENs mainlycomprise a highly specific DNA-binding domain derived from TALE and anon-specific endonuclease domain of Fok I. TALENs can recognize and bind specificDNA sequences to generate double-strand breaks (DSBs). DSBs can stimulate therepair mechanisms that are dependent on intracellular non-homologous end joining(NHEJ) and homologous recombination(HR). These two repair mechanisms canfacilitate the occurrence of gene knockouts or knock-ins in the genome.In this report, we used the efficient gene targeting technology TALEN for thetargeted modification of the CCR5gene in rabbits and albumin gene in pigs. Weexplored the feasibility of generating an HIV-1model by knocking in human CD4andCCR5gene coding sequencing (CDS) into rabbit genome. We also constructed ananimal model for expressing human serum albumin in the blood of pigs by knockingin human albumin gene CDS into pig genome.We constructed two TALEN vectors targeting the rabbit CCR5gene and a vectorwith homologous arms. TALEN mRNAs and donor DNA were then co-injected intofertilized oocytes. After3–5days,24embryos were collected and used for PCR andsequencing in mutation analysis. All embryos were detected with CCR5knockouts,among which five were human CD4and CCR5knock-ins. This finding indicates thepossibility of obtaining human CCR5and human CD4knocked in rabbits using thismethod. Thereby, the microinjected fertilized oocytes were then transferred into thesurrogate female rabbits. We expected the newborn rabbits with knocked in humanCD4and human CCR5in the rabbit CCR5gene locus. Our results provide thefoundation for establishing a new animal model for the study of AIDS. We constructed one TALEN vector targeting the pig albumin gene and a vectorwith homologous arms. TALEN vectors and homologous-targeting vector weretransfected into porcine fetal fibroblast cells using electric transfection. Aftertransfection and selection via puromycin, we obtained two positive cell lines withknocked in human albumin gene CDS in the pig albumin gene locus. The two positivecell lines were used as donor cells for nuclear transfer, and then transferred into thesurrogate female pigs. After about114days of pregnancy, we obtained two positiveknock-in pigs. We extracted albumin from the blood of positive pigs for massspectroscopy analysis, and detected the expression of human serum albumin. Ourresults provide the foundation for establishing a new animal bioreactor model toexpress human serum albumin.