| Modification of cultured cells combined with somatic cell nuclear transfer (SCNT) provides an effective approach to obtain large transgenic animals. Genetically modified domestic animals may play an important role in agriculture and biomedical research. Mini-pig breeds are approximately the same size as humans, their metabolism and the structure of their organs are similar; the pig genome is much closer to that of humans than the mouse genome. Accordingly, pigs are regarded as primary candidates for human disease model for xenotransplantation until recently:the efficiency of porcine clone is low. The aim of this study was to optimize the condition of production of transgenic cloned embryosThe methods and results of the experiments were summarized as follows:1. Isolation and culture of somatic cells in bama Miniature Pig.In this experiment, ear Fibroblast cells, kidney fibroblast cells and testicle fibroblast cells were obtained from a new born bama Miniature Pig by using a primary explant technique. The three type cells were identified by morphology and Immunofluorescence.The result indicated that the vimentin was positive and the Angle protein was negative and according the cell’s morphology and the source of tissue, we successfully established ear fibroblast cells, kidney fibroblast cells and testis fibroblast cells.2. Study on establishment of transgenic cell line and their biological character.To estabilish a high quality transgenic positive cell line is the key to obtain transgenic cloned animals. The experimental results were as follows. Positive cells were obtained after plasmids were transformed into (kidney, ear and testicle) cells with lipofectamineTM 2000, by bleomycin selecting.The PCR identification results were consistent with the purpose fragment size. No contamination of microorganisms were observed in the culture media after testing the three transgenic cells line for bacteria,fungi,viruses and mycoplasmas. No viruses were indicated by the hemadsorption test. After staining with Hoechst 33342, fibroblast nuclei appeared as blue ellipses under a fluorescence microscope, and the PCR showing that the established cell line was mycoplasma negative. The result of karyotype analysis of different passages showed that the rate of normal karyotype reduced with the increase of the generation time, but the difference was not significant (p<0.05). In a long-term culture experiment, the kidney transgenic cells were maintained for a high number of passages at least up to passage 56, transgenic ear cells up to passage 30 and Testicular transgenic cells up to 25. In conclusion:in this study three transgenic cell lines were successfully established with the high quality and sufficient donor cells for the subsequent somatic cell nuclear transfer.3. Optimization of fusion and activation and constraction of transgenic cloned embryosFusion/activation is a key step of reconstructing somatic cell nuclear transfer embryos and only the embryos were fused/activated that have been fused/activated can start the gene expression and continue to develop. In this experiment we optimized the activation parameters suitable for transgenic cloned embryos fusion and the results were as follows. Pulse in 30 us, one pulse, the field strength of 1.5 kv/cm group,the fusion rate (87.32%) and cleavage rate (69.79%) was significantly higher than 1.0 kv/cm group(78.63%,45.07%)and 2.0 kv/cm group(63.87%,36.72%). The blastocyst rate was significantly higher than 1.0 kv/cmand 2.0 kv/cm (21.74%vs.9.65%,49%, p< 0.05); when the pulse for 30 us, the fusion rate (81.92%) and blastocyst rate (19.56%) is significantly higher than 20 us (69.19%,9.19%) and 40 μs (69.28%,6.51%) (p< 0.05); the fusion rate (63.32%) and cleavage rate (38.09%) and blastocyst rate (5.49%) of the three times pulse group were significantly lower than 1 pulse (81.92%,63.40%,33.15%) and 2 pulses (81.59%,67.46%,12.97%). In the same pulse frequency and pulse time, transgenic cloned embryos need higher field strength than the no-transgenic embryo. In conclusion, the most suitable fusion activation parameters (1.5 kv/cm,30μs,1 pulse) for production of transgenic cloned embryos are optimized.4. Effect of cell cycle synchronization, cell types and cell passages of donor cells on the development of transgenic cloned embryos importantCell cycle synchronization, cell types and cell passages of donor cells are important factors for successful animal cloning by nuclear transfer. In this study, we evaluated the effects of serum starvation, roscovitine and serum starvation on the transgenic cells cycle synchronization.We studied the effects on the developmental potency of embryo by using the treated transgenic cells as donor cell for somatic cell nuclear transfer. We compared the embryo development potency of different kinds of transgenic cell as donor cells and different passages cells of the donor. The result showed that the percentage of GO/G1 phase cells in contact inhibition group and roscovitine treatment group was significantly higher than that in serum starvation group (92.11%,89.59%vs.80.82%, p<0.05). A significantly higher rate of apoptosis was occurred in serum starvation group than in contact inhibitionand roscovitine treatment groups (12.57%vs.6.71%, 2.46%, p<0.05). There was a significant decrease in blastocyst yield in serum starvation group compared to roscovitine treatment group and contact inhibition group (14.19%vs.1.31%,20.32%, p<0.05), however, no significant difference was found between roscovitine treatment and contact inhibition groups.There were no significantly differences of blastocyst rate (18.08%,10.19%,10.76%, respectively, p>0.05) of the transgenic kidney, ear and testicles cell as donor cell. Using the transgenic kidney cells passage 10,20 and 30 as donor cell for SCNT, the balstocyst rate is no significant (P>0.05).In conclusion, the above clearly indicate that Bama mini pig transgenic kidney fibroblasts could be effectively synchronized at the GO/G1 stages by the three different treatments. The cells treated by roscovitine and contact inhibition could be used as nuclear donors and improve development of transgenic cloned embryo, three types of genetically engineered cells all support the development of cloned embryos, the blastocyst rate is highest using transgenic kidney fibroblasts as donor cell, kidney cells are more suitable for production of transgenic porcine clone by using somatic cell nuclear transfer (SCNT).5. Effect of oxamflatinon development of transgenic cloned embryos.The faulty epigenetic reprogramming of somatic nuclei is the main cause of low cloning efficiency. Several studies reported that by adding the histone deacetylase inhibitors (HDACi) such as oxamflatin could enhance the development of somatic cell nuclear transfer embryos. In the present study, we examined whether oxamflatin ccould promote the developmental competence of SCNT embryos. Our results showed that 1μM oxamflatin for 12h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from transgenic kidney fibroblast cells compared to the control(29.63%vs.17.53%, p<0.05). We found that the acetylation level of Histone H41ysine8 in 2-cell and blastocysts and the express level of pluripotency-related genes Oct4, Sox2, Nanog and anti-apoptotic gene Bcl-2 in SCNT blastocysts were significantly higher than that with the control group (p<0.05), however, the expression of the pro-apoptotic gene Bax reduced, the oxamflatin could increase the in vitro developmental competence. In conclusion, Oxamflatin improves embryonic development and alters gene expression in pigs subsequently enhances the nuclear reprogramming and developmental potential of SCNT embryos.6. Embryo transfer and identification of cloned pigAfter embryo transfer, one piglet is positive idensfied by using PCR, under the fluorescence microscope observation and using the western blotting technology. |
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