Establishment Of Cloned Minipigs Models For Congenital Nephrotic Syndrome By Somatic Cell Nuclear Transfer

Objective: Congenital nephrotic syndrome(Congenital Nephrotic, Syndrome, CNS)(Finland) features: At approximately 3 months after birth showed typical manifestations of nephrotic syndrome. In most cases, the children with the disease are symptoms during in utero, manifested as low birth weight, can rapidly deteriorate to glomerular sclerosis stage in a very short period of time, hormone and immune suppression of traditional therapy, most patients dead of serious complications in about 6 months after birth. The disease gene is NPHS1(located in the long arm of human chromosome 19- 13.1). As a human in the pathology and disease have highly similarities animal, experimental miniature pigs has the very high value of disease modeling. Transcription activator effector nuclease(transcription activatorlike effector nuclease, TALENs) as a multi species common gene knockout technology, the pig fetal fibroblast cells were accurate gene modification. "The technology of somatic cell nuclear transfer"(somatic-cell-nuclear transfer, SCNT) refers to: "the mammalian early embryonic cells(early embryonic cells)" or "somatic cell nuclei into" artificial "to the nuclei(denucleate)" of the fertilized eggs or oocytes, genotype of offspring of the final delivery and the same donor cells, this process is also known as "cloning(Clone) or" asexual reproduction(asexual reproduction). We use the gene modified cells obtained by the TALENs technique in the experiment(NPHS1 biallelic knockout pig fetal fibroblast cells as donor nuclei) source of clones, with the help of SCNT to create NPHS1- /- congenital nephrotic syndrome of cloned pigs, disease animal model for future research.Methods: 1)The "Kinmen method(Golden Gate)" for the construction of NPHS1 exon 2(exon 2) of the TALENs vector, and then the "electric shock nucleofection method" TALEN plasmid and screening of resistance plasmid was transfected into porcine primary fibroblasts(Porcine primary fibroblasts, PPF) NPHS1- /-cells. 2)The NPHS1gene modified pigs to fetal fibroblast as donor(after recovery culture and suspension liquid), with porcine oocytes during maturation in vitro. 3) Applying "blind aspiration(Blind Aspiration method)", and discard the egg nucleus, partial perivitelline injection of NPHS1- /- cells. 4)The use of "electric pulse stimulation(electric pulse stimulation)", will be a step to get the "embryos" fusion operation. 5)The stereo microscope observation, judgment whether the donor cells and oocytes of effective integration, pick up the non fusion and fusion rate of reconstructed embryos(embryonic fusion is activated at the same time). 6)The short-term in vitro embryo culture(In vitro embryo culture), operation methods and embryo transfer receptor into surrogate sows. 7)To maintain the surrogate sows was 115 ± 2 days of pregnancy, during pregnancy ultrasound judgment. 8)The tissues were cloned piglets after delivery, the pig genome, using PCR- sequencing to identify the positive cloned piglets.Results: 1)The TALEN target was screened after NPHS1- /-cells in the perivitelline injection of NPHS1- /- cells。2) Statistics of each batch of electric pulse fusion rate was good. 3)The selected part of another operation embryo transplantation recipients, the cumulative 5 sows, successfully into the embryos were 470, 460, 410, 380, 370, and 30 days of pregnancy ultrasound in the diagnosis of the 3 Statistics, the conception rate is 60%. 4)The 2 of them head pregnant receptor in pregnancy 90 days about the embryo degeneration and absorption, the other 1 in piglets pregnant 117 days produced 1 clones. The length of cloned piglets, pigs were similar with the natural volume, temperature fluctuations within the normal range for milk, similar in normal activities, with similar age, and showed a good ability to adapt to the outside world. 5)The cloned piglets tissues and extract genomic PCR- sequencing for the wild type(wild type, WT).Conclusion: We use TALENs gene targeting and shock the nucleofection technique(Electric nuclear transfection) were successfully obtained NPHS1 "biallelic knockout" PPF, confirmed by TALEN knock-out porcine fetal fibroblasts can be better by receptor selection pressure. We choose the NPHS1 gene knock out the perivitelline space inof high fusion rate, confirmed the method used in this experiment and the parameters is feasible, can obtain stable results also confirmed that the cloned gene modified cells; TALEN experience in vitro selection pressure, fully able to satisfactorily as a source of donor cells for nuclear transfer. We successfully use embryo transplant recipients in early pregnancy rate of sows, reached the level of 60%, pre pregnancy NPHS1 targeting cloning embryos and "clone" rate at the same level, that the nuclear route transfer technology and operating parameters used in this study is feasible. Also confirmed that TALEN targeting PPF cells as donor cells, a programmable full "(reprogramming)" and obtain a higher pregnancy rate. In this experiment, although the cloned pigs, identified only the wild type WT, the cloning is not able to successfully NPHS1 gene knockout pigs, not the successful construction of congenital nephrotic syndrome model, to be re somatic cell nuclear transfer.