Preliminary Studies On Issues Related To Guangxi Bama Mini-Pig Somatic Cell Nuclear Transfer And Monkey-Pig Interspecies Somatic Cell Nuclera Transfer

The aims of this study were to investigate issues related to Guangxi bama mini-pig somatic cell nuclear transfer(SCNT)and monkey-pig interspccies somatic cell nuclear transfer.This thesis was arranged with two parts.The first part was dealt with literature review and the second part was with regard to the experimental studies which included:(1)establishment of somatic cell line from Guangxi Bama mini-pig and monkey;(2)establishment of system of Guangxi bama mini-pig somatic cell nuclear transfer;(3)Effect of PHA on parthenogenetic activation and cloned embryo development;(4)establishment of method for transfer of cloned embryos in pig;(5)primary investigation on monkey-pig interspecies somatic cell nuclear transfer.Summaries of this study could be made as the followings.1.Four somatic cell lines were set up from Guangxi Bama mini-pig testicle fibroblast cells and ear fibroblast cells;pig fetal fibroblast cells and granulose cells.It was also investigated whether these cells could be used as donor cells for SCNT.Cells from a new-born Guangxi Bama piglet were isolated and cultured by enzyme-digesting and tissue-piece methods and their cell types were identified by fluorescent immunocytochemistry(ICC).Furthermore,the relationship between cell cycle synchronization and nucleus histone acetylation levels were also investigated.It was concluded that the culture system was good enough for these fibroblast cells.The result of(ICC)indicated that isolated cells from testicle tissues were testicle fibroblast cells.The cells were synchronizated by serum starvation or contact inhibition.With the extending of treatment time in serum starvation group,synchronizated G0/G1 cells inereased rapidly and then kept steady after 2d.Synchronization effiency were sighnificantly higher in treated 2d,4d group than 70-80%conflence group(75.9%,95.9%vs.95.2%, P＜0.05);Similar result was also obtained in contact inhibition group. Synchronization efficiency were sighnificantly higher in 2d,4d and 6d contact inhibition group than the 70-80%and 100%conflence group(97.3%,95.0%, 97.4%vs.74.7%,84.2%,P＜0.05).The level of nucleus histone acetylation in G0/G1 donor cell was similar trend with different treated time by serum starvation or contact inhibition.The level of nucleus histone acetylation increased to the highest level at 2d,then decreased at lowest level.2.Long-tailed macaque ear fibroblast cell was isolated and cultured in vitro and whether it could be used for donor cells in intra-species and inter-species cloning was also investigated.Fibroblast cells were isolated and cultured by enzyme-digesting and tissue-piece methods.Cell type was also identified by fluorescent immunocytochemistry(ICC)and the result indicated that isolated cells were fibroblast cells.The result of chromosome analysis showed that ear fibroblast cells still kept normal when cultured up to passage 21.Now ear fibroblast cells have been passaged 43 times,but still proliferate well.3.The purpose of this experiment was the followings:A.to investigate the moving rule of the first polar body in oocyte maturation so as to improve enucleation efficiency.B.to investigate the development capacity cloned embryos from fetal fibroblast cells and granulose cells.C.to investigate the effect of insulin on development of pathenogenetic and cloned embryos.D. to investigate the development capacity of parthenogenetic and cloned embryos using simple fusion equipment.Results showed that after the oocyte maturation for 44-46h,84.7%polar body moving-angle aren't beyond 30°.During this time 88.1%oocytes were successfully enucleated by blind-enucleation method;there was no significantly positive effect of insulin on the development of pathenogenetic(47.3%vs.44.3%,P＞0.05)and cloned embryos(8.0%vs.2.4%, P＞0.05);for the simple fusion equipment,the morula rate of cloned embryos was a litter higher in 200v/mm group than 220v/mm group,although there were no significant difference(33.3%,29.6%vs.17.1%,13.8%,P＞0.05);20μs pulse duration group was a little higher than 40μs group when used the same field strength.It indicated that as the field strength and pulse duration increased,the development rate of morual fell down;for pathenogenetic embryos,the blastocyst rate at 20μs group was a little higher than that at 40μs group when 200v/mm field strength was used,but there was no significant difference.4.the optimal cell cycle synchronization protocol of new-born Guangxi Bama mini-pig testicle fibroblast cell(passage3-5)was investigated by contact inhibition and serum starvation;Bama mini-pig testicle fibroblast cell (passage10-14)was introduced into the enucleated oocytes to investigate whether it could be reprogrammed after activation;to determine the potential of the testical fibroblast cells as donor cell,the in vitro developmental capacity of the cloned embryos were examined using low(3-5)and high passage(10-14) testical fibroblast cells,compared to ear fibroblast cells.Result showed that the fusion rate was significantly higher in contact inhibition group than that in starvation group(68.6%vs.55.3%,P＜0.05).The rate of cleaved embryos was similar between the two groups(83.8%vs.82.8%,P＞0.05),while development rate to the blastocyst was higher in contact inhibition group than that in the starvation group(21.1%vs.14.1%,P＞0.05);At 3 hours after activation,the nucleus was clearly visible and with the same size as the donor cell. Enlargement of the nucleus was investigated at 6h after activation.At 12h after activation the number of the reconstructed embryos with enlargement nucleus increased,and some of them even cleaved;there was significant difference in fusion rate using either ear fibroblasts or low(3-5)and high passage(10-14) testicular fibroblasts(84.4%,79.1%vs.69.2%,P＜0.05).Similar cleavage rates were found in all three groups.Low passage testicular fibroblasts resulted in the highest rate of blastocyst production but there was no significant difference between high passage testicular fibroblasts and ear fibroblasts.5.The effect of PHA in culture medium on parthenogenetic and cloned embryos development was examined.Result showed that parthenogenetic embryo development to the blastocyst stage in the presence of PHA(10μg/ml)was higher than in control group(0μg/ml),but there was no significant difference; however,the percentage of cloned embryo development developing to the blastocyst stage in the presence of PHA(10μg/ml)was significantly higher than that with treatment group(20μg/ml)and control group(0μg/ml)(13.0%vs.5.6%, 5.0%,P＜0.05).In a word,the influence of PHA on parthenogenetic and cloned embryo was dose-independent.Treatment of 10μg/ml PHA is the optimal concentration to sustain cloned and parthenogenetic embryos development.6.In order to evaluate the development capacity of cloned embryo in vivo,the reconstructed embryos from fetal fibroblast cells and bama mini-pig testicle fibroblast cells were penetrated into the oviducts of the surrogate gilts with a needle.A total of 4 surrogate gilts were used and two of them were bama min-pig and the other two were Guangxi Luchuan pig.Result showed that both two Bama mini-pig surrogate returned to estrus.One of the two luchuan gilts gave birth to one healthy male Bama mini-pig.The cloned embryos using Bama mini-pig testicle fibroblast cells as donor cell could develope to term in vivo.7.Long-tailed macaque ear fibroblast cells,as donor cells,were introduced into pig enucleated oocytes to investigate whether they could be reprogrammed. To investigate appropriate embryo culture conditions for interspecies cloned embryos,three culture mediums were used:NCSU-23,NCSU-23+10%FCS, TCM199+10%FCS.Results showed that 6h after activation,the introduced nucleus gradually became swollen,which indicated that monkey ear fibroblast cells could be reprogrammed.The fusion rate of monkey-pig reconstruct embryos was 74.2%;70.5%of reconstructed embryos cleaved and 29%cleaved embryos developed to morula;all the three culture media could help the reconstructed embryos break through 4-cells block and develop to beyond 8-cells(23.5%,17.0%vs 19.9%,P＞0.05).And a few reconstructed embryos developed to blastoeysts(1.4%vs.1.1%,P＞0.05)in media of NCSU23+10%FCS,and TCM199+10%FCS,but there was no significant difference between these two media.