Genetic modified pig production with combination of various gene modulating technologies

Abstract

The most ideal way to produce cloned pigs to date is SCNT. Due to comparative simplicity of overexpression than gene KO, lots of target gene overexpressed pigs have been produced while less KO pigs were reported. Recently, diverse genetic scissors (i.e., ZFN, TALEN or RGEN), which rapidly increase the number of KO animals including pigs have been started to highlight. Pigs are considered to be an important large animal model in biomedical research. Due to absence of porcine ESCs and characterized cell lines, development velocities of genetic modification technologies and transgenic pig production are tardy. In this study, to overcome absence of characterized cell lines, hTERT genes overexpressing immortalized cell lines were established and characterized. These cells were further used to apply genetic modification technology (i.e., KO by TALEN). The gene overexpression and KO technologies were respectively applied for genetic modified pig production (i.e., Neurodegenerative disorder pig model). For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the hTERT transfection. After selecting cells with neomycin for two weeks, outgrowing colonized cells were picked up and sub-cultured for expansion. Various analyses were accomplished and characterized for confirming target gene insertion in cells. Immortalized cells were cultured for more than 9 months without changing their doubling time (approximately 24 h) or their diameter (< 20 μm) while control cells became senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to KO the CMAH gene, designed plasmids encoding a TALENs pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation sensitive T7E1 assay, fPCR and sequencing to obtain 3 independent clonal populations of cells that contained biallelic modifications. The GGTA1 gene KO processes was exactly same with that of CMAH KO cell line establishment method. One hTERT overexpressed plus CMAH KO clone was chosen and used for SCNT. Cloned embryos developed to the blastocyst stage. To produce target gene overexpressed and knocked out pigs, PD related genes were selected

SNCA and PRKN. Here, I produced 23 cloned pigs expressing human SNCA via SCNT. Among them, 4 pigs survived (designated PDF4, PDF16, PDF18 and PDF20) and were confirmed as transgenic animals by PCR based analysis. Target proteins were detected in only 2 transgenic pigs (PDF4 and PDF18) by ELISA at 4 months of age. However, SNCA concentrations in blood samples were changed at 1 year of age. PDF4 and PDF20 showed significantly increased levels of SNCA. The SNCA concentration in PDF18 significantly decreased, while that of PDF16 did not change during 1 year after birth. Behavior scoring analysis was carried out in control pigs and all cloned pigs at timed intervals (Control, unchanged

PDF18, decreased

PDF4, PDF16 and PDF20, increased

PD like behavioral changes lead score increasing). Similar changes were detected in both ELISA and behavior scoring analyses at timed intervals. PRKN gene was chosen for KO pig production. PRKN specific TALEN pairs were transfected in to pig somatic cells with reporter, 2 to 3 days after transfection GFP signal released cells were chosen for SCNT without specific selection processes. Eight cloned piglets were born and named, PDM1 to 8. In three (PDM3

heterozygote, PDM5

homozygote, PDM6

homozygote) out of eight, PRKN mutation was confirmed. However, these three KO piglets were dead (PDM3

unknown reason, PDM5

euthanasia because of cleft, PDM6

stillbirth). Nevertheless cell lines from those KO pigs except PDM6 were established and will be employed for re-cloned KO piglets. In conclusion, I demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene and the TALEN pairs enabled gene disruptions in these immortalized cells in vitro. Additionally, these transfected cells also develop into the blastocyst stages via SCNT. Based on these in vitro studies, SNCA overexpressed pigs and PRKN KO pigs were successfully produced. Furthermore protein analysis, behavioral changes, and brain imaging analysis were scheduled in both SNCA overexpressed pigs and PRKN KO pigs those were planned to reproduce.