Studies On Somatic Cell Cloning And Genetic Modification In Guangxi Unique Native Miniature Pig Breeds

Bama minipig and Huanjiang Xiang pig are two unique miniature pig breeds originating from Bama Yao Autonomous County and Huanjiang Maonan Autonomous County of Guangxi,China,respectively.These two Guangxi unique native miniature pig breeds have significant value in the fields of agriculture and biomedicine.This thesis was focused on the application of somatic cell cloning and somatic cell cloning mediated genetic modification in Bama minipigs and Huanjiang Xiang pigs,laying a foundation for genetic resources preservation,propagation of superior breeding pigs,genetic breeding of these Guangxi unique native miniature pig breeds.In addition,this thesis also lays a foundation for generating somatic cell cloned and genetically modified Guangxi unique native miniature pig breeds with practical applications in agriculture and bio-medicine.The main works and results of this thesis are summarized as follows:(1)Production of somatic cell cloned Bama minipigs and construction of transgenic cloned embryosThis study was aimed to build the technical system for Production of somatic cell cloned Bama minipigs and construction of transgenic cloned embryos.Firstly,kidney fibroblast cells were established from one newborn male Bama minipigs.The kidney fibroblast cells showed excellent cell proliferation activity and were able to support completely developmental potential of cloned embryos in vitro and in vivo and eventually received a cloning efficiency as 0.66%(11/1658),indicating the neonatal pig kidney fibroblast cell is a suitable somatic cell resource for production of somatic cell cloned Bama minipigs.After then,Xfect transfection reagent and a red fluorescent protein,DsRed,transgenic plasmid were employed to test whether the newborn pig kidney fibroblast cell is suitable for genetic modification.Result showed that,under the optimized transfection conditions(DNA dose and Xfect reagent ratio was 5.0 ?g/1.5 ?l;with incubation at 12 h),the transfection efficiency was 16.6 ± 0.6%.Genetically modified cells showed normal cellular morphology and exuberant proliferation activity.Genetic modification didn't damage the in vitro developmental potential of subsequent cloned embryos,and intense expression of exogenous DsRed had also been detected in all in vitro developmental periods of cloned embryos.(2)Generation of transgenic-cloned Huanjiang Xiang pigs systemically expressing enhanced green fluorescent proteinHuanjiang Xiang pig is a unique native minipig breed originating in Guangxi,China,however,its somatic cell cloning and genetic modification has not been reported.This chapter was aimed to generate transgenic-cloned Huanjiang Xiang pig,basing on the technological system used for generating somatic cell cloned and transgenic-cloned Bama minipigs.Firstly,skin fibroblast cells were established from one newborn male Huanjiang Xiang pig.Transgenic fibroblast cells expressing enhanced green fluorescent protein(eGFP)were established using Xfect reagent.After then,cloned embryos and transgenic-cloned Huanjiang Xiang pig embryos expressing eGFP in all in vitro developmental periods were produced by SCNT.The effect that scriptaid,a widely-used histone deacetylase inhibitor,could enhance the in vitro development of porcine cloned embryos had also been reproduced in Huanjiang Xiang pig cloned and transgenic-cloned embryos.Finally,three live somatic cell cloned Huanjiang Xiang pigs carrying exogenous eGFP were generated after embryo transfer.Overall cloning efficiency was 0.6%(3/504).In addition,expression of eGFP was detected from various organs or tissues from positive transgenic-cloned Huanjiang Xiang pigs.Transgenic-cloned Huanjiang Xiang pigs have been successfully generated in this study.(3)Generation of transgenic-cloned Bama minipigs carrying hGFAP-DsRed transgeneThis study was aimed to utilize human glial fibrillar acidic protein(hGFAP)promoter to generate transgenic-cloned Bama minipigs with astrocytes-specific expression of DsRed,providing a large animal model for astrocytes-related researches.Transgenic kidney fibroblast cells carrying hGFAP-DsRed transgene were firstly established and were used as donor cells for production of cloned embryos.By using a practical strategy that significantly increases the number of transferred embryos during embryo transfer,we finally successfully obtained live somatic cell cloned Bama minipigs.Somatic cell cloned Bama minipigs remained clinically healthy and showed normal growth and development.Genotype identification showed the exogenous hGFAP-DsRed gene fragment integrated into the genome of living somatic cell cloned Bama minipigs.Histological identification showed the astrocytes-specific expression of DsRed in positive transgenic-cloned Bama minipigs.(4)Generation of Bama minipigs harboring mutations in ?-synuclein causing Parkinson's diseaseThis study was aimed to generate Bama minipigs harboring mutations in?-synuclein causing Parkinson's disease(PD)via somatic cell cloning combining with CRISPR/CAS9 mediated gene editing,providing a large animal model for PD research.Kidney fibroblast cells carried monoallelic three mutations(E46K/H50Q/G51D)in ?-synuclein were firstly established and were identified without exogenous genes integration and off-targets.After then,these cells with precision site-specific modification were used to produce somatic cell cloned piglets.Nine live somatic cell cloned piglets were obtained.Eight of these somatic cell cloned piglets were identified as carrying desired mutations by genotyping.Mutant minipigs remained clinically healthy and showed normal growth and development.However,when they reached at 3-month-old,non gene editing Bama minipigs appeared PD-like symptoms and pathological changes.Considering PD is closely related with age,such mutant Bama minipigs should be continuously examined in the occurrence of PD-like pathological features which may finally validate the use of this large animal model in PD research.In summary,this thesis lays the foundation for utilizing somatic cell cloning technique in genetic resources preservation of Guangxi unique native miniature pig breeds and in rapid propagation of superior breeding pigs.In addition,this thesis also lays the foundation for utilizing somatic cell cloning mediated genetic modification technique in the generation of genetically modified Guangxi unique native miniature pig breeds with practical applications in agriculture and bio-medicine.