山羊胎儿成纤维细胞的培养鉴定及其转基因核移植效率的研究
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摘要
实验一:本实验主要利用了SRY-PCR技术对四株奶山羊胎儿成纤维细胞系进行了性别鉴定,并检测了细胞周期抑制剂roscovitine,DMSO对山羊供体细胞的同期化作用,以确定它们是否可用于山羊供体细胞的同期化处理。SRY-PCR结果显示,有三株细胞为雌性细胞,一株细胞为雄性细胞。同期化检验的结果表明,与传统的血清饥饿法相比,阻断药物的处理都显著提高了供体细胞的同步化程度。其中15uM roscovitine处理48h显著增加了G0+G1期细胞比例(87.70%),但G0期细胞比例并未显著增加(6.84%)。1% DMSO处理48h诱导32.54%的细胞进入G0期。TUNEL细胞凋亡法分析发现,与血清饥饿法处理相比,周期阻断药物roscovitine(1.42%),DMSO(0.9%)与正常增殖的细胞(1.12%)相比并没有增加凋亡率。这表明SRY-PCR技术可运用于成纤维细胞的性别鉴定,roscovitine和DMSO也可用于核移植过程中的供体细胞同期化处理。
实验二:本实验主要检测了四株不同个体来源的胎儿成纤维细胞GFF1, GFF2, GFF3和GFF4对外源基因的转染效率,以及对核移植重构胚发育能力的影响。利用四株胎儿成纤维细胞作为供体细胞进行核移植,并对重构胚囊胚率进行比较。实验结果表明,利用四组胎儿成纤维细胞作为供体细胞,其重构胚囊胚率存在显著差异(16.46±0.42%, 16.13±1.26%, 17.02±1.54% VS 7.69±0.28%.)。对四株胎儿成纤维细胞进行PEGFP-C1转染,G418筛选后比较转基因阳性细胞克隆数和可扩增的阳性细胞克隆数,实验结果表明,GFF1和GFF2的G418阳性克隆数和可扩增单克隆细胞数目都显著高于GFF3和GFF4(P<0.05)。实验结果表明,供体细胞的遗传背景,对核移植重构胚发育能力,以及对外源基因的转染效率的也有所影响。
实验三:为了优化山羊核移植胚胎体外培养体系,提高核移植效率,本研究检测了山羊体细胞核移植(SCNT)胚胎在序贯培养液G1/G2中的发育率和囊胚细胞凋亡,以及核移植胚胎移植后的妊娠率,以传统mSOF-FBS培养液作为对照组,评估序贯培养液G1/G2支持山羊核移植胚胎的发育能力。结果显示,G1/G2组的囊胚发育率与对照组差异不显著(27.7±3.1% vs 25.3±1.0%,P>0.05);囊胚细胞数和囊胚细胞凋亡率mSOF-FBS组显著高于G1/G2组(分别为109.1±6.2 vs 93.2±4.5和11.3±0.1% vs 4.9±0.2%,P<0.05),但移植后的妊娠率G1/G2组显著高于mSOF-FBS组(21.4% vs 8.0%,P<0.05)。结果表明,与传统的培养液mSOF-FBS相比,序贯培养液G1/G2能更好地支持山羊核移植胚胎的发育。
Experment 1:In this present study, we used SRY-PCR in cell gender identification, and investigate effects of cell cycle inhibitors: roscovitine and DMSO on the goat fetal fibroblast cells. The result of SRY-PCR indentified 3 female cell lines ,and 1 male lines. After the treatment of15uM roscovitine for 48 h, the percentage of fibroblast cells arrested in G0/G1 phases was 87.70%, but the percentage of cells arrested in G0 phases is not significantly increased (6.84%). 1% DMSO for 48h could arrests 32.54% fibroblasts in G0 phases. The results of TUNEL assay show that compared with the control group(1.12%), cell cycle inhibitors did not induced higher rates of cell apoptosis(roscovitine 1.42% , DMSO 0.9%).These results indicated that the SRY-PCR could be used in cell gender identification, and the use of cell cycle inhibitors may be a more effective and healthy in synchronization of donor cells.
Experment 2: The present study was to investigate the differences of donor cell lines derived from the same tissue but different animal sources to support development of cloned embryos , and the differences in the efficiency of transfected exogenous gene on the developmental competence of goat SCNT embryos. Four cell lines isolated respectively from four goat fetuses were used as donor cells in SCNT. The development of SCNT embryos show a significant difference among the four groups in blastocyst rates(16.46±0.42%, 16.13±1.26%, 17.02±1.54% VS 7.69±0.28%.) . This four fetal fibroblasts are transfected with PEGFP - C1, positive cell clones and amplified positive cell clones are compared after G418 screening. The results show that the number of positive clones and amplified cell clones of GFF1 and GFF2 are significantly higher than GFF3 and GFF4 (P < 0.05). The two strains chosen from GFF1 clones are used as donor cells, and compared by GFF1, there is no significant difference in blastocyst rates among three groups. The results show that different sources of the fetal fibroblasts also effect the development of cloned embryos ,and the efficiency of transfected exogenous gene on the developmental competence of goat SCNT embryos.
Experment 3: The present study was to investigate effects of sequential media G1/G2 and modified synthetic oviductal fluid (mSOF) culture systems on developmental competence, cell apoptosis and the pregnancy of goat somatic cell nuclear transfer (SCNT) embryos. Goat NT embryos were cultured as following: (1) 72 h in G1 then 120 h in G2.3 (group G1G2), both the culture media supplemented with 0.8% BSA (bovine serum albumin); (2) 72 h in mSOF supplemented with 0.8 % BSA then in mSOF supplemented with 10% FBS (fetal bovine serum) for 120 h (group mSOF-FBS). The results indicated that there was no significant difference between the sequential media G1/G2 and mSOF-FBS in blastocyst rates (27.7±3.1% vs 25.3±1.0%,P>0.05). Both numbers of total cell and apoptotic nuclei in blastocyst from G1/G2 were significantly higher than mSOF-FBS (109.1±6.2 vs 93.2±4.5 and 11.3±0.1% vs 4.9±0.2% respectively, P<0.05 ). However, the pregnant rates on Day 30 in group G1/G2 were higher than those in group mSOF-FBS(21.4% vs 8.0%, P<0.05 ). In conclusion, compared to mSOF-FBS, the sequential media G1/G2 could better support the development of goat somatic cell nuclear transfer embryos.
实验二:本实验主要检测了四株不同个体来源的胎儿成纤维细胞GFF1, GFF2, GFF3和GFF4对外源基因的转染效率,以及对核移植重构胚发育能力的影响。利用四株胎儿成纤维细胞作为供体细胞进行核移植,并对重构胚囊胚率进行比较。实验结果表明,利用四组胎儿成纤维细胞作为供体细胞,其重构胚囊胚率存在显著差异(16.46±0.42%, 16.13±1.26%, 17.02±1.54% VS 7.69±0.28%.)。对四株胎儿成纤维细胞进行PEGFP-C1转染,G418筛选后比较转基因阳性细胞克隆数和可扩增的阳性细胞克隆数,实验结果表明,GFF1和GFF2的G418阳性克隆数和可扩增单克隆细胞数目都显著高于GFF3和GFF4(P<0.05)。实验结果表明,供体细胞的遗传背景,对核移植重构胚发育能力,以及对外源基因的转染效率的也有所影响。
实验三:为了优化山羊核移植胚胎体外培养体系,提高核移植效率,本研究检测了山羊体细胞核移植(SCNT)胚胎在序贯培养液G1/G2中的发育率和囊胚细胞凋亡,以及核移植胚胎移植后的妊娠率,以传统mSOF-FBS培养液作为对照组,评估序贯培养液G1/G2支持山羊核移植胚胎的发育能力。结果显示,G1/G2组的囊胚发育率与对照组差异不显著(27.7±3.1% vs 25.3±1.0%,P>0.05);囊胚细胞数和囊胚细胞凋亡率mSOF-FBS组显著高于G1/G2组(分别为109.1±6.2 vs 93.2±4.5和11.3±0.1% vs 4.9±0.2%,P<0.05),但移植后的妊娠率G1/G2组显著高于mSOF-FBS组(21.4% vs 8.0%,P<0.05)。结果表明,与传统的培养液mSOF-FBS相比,序贯培养液G1/G2能更好地支持山羊核移植胚胎的发育。
Experment 1:In this present study, we used SRY-PCR in cell gender identification, and investigate effects of cell cycle inhibitors: roscovitine and DMSO on the goat fetal fibroblast cells. The result of SRY-PCR indentified 3 female cell lines ,and 1 male lines. After the treatment of15uM roscovitine for 48 h, the percentage of fibroblast cells arrested in G0/G1 phases was 87.70%, but the percentage of cells arrested in G0 phases is not significantly increased (6.84%). 1% DMSO for 48h could arrests 32.54% fibroblasts in G0 phases. The results of TUNEL assay show that compared with the control group(1.12%), cell cycle inhibitors did not induced higher rates of cell apoptosis(roscovitine 1.42% , DMSO 0.9%).These results indicated that the SRY-PCR could be used in cell gender identification, and the use of cell cycle inhibitors may be a more effective and healthy in synchronization of donor cells.
Experment 2: The present study was to investigate the differences of donor cell lines derived from the same tissue but different animal sources to support development of cloned embryos , and the differences in the efficiency of transfected exogenous gene on the developmental competence of goat SCNT embryos. Four cell lines isolated respectively from four goat fetuses were used as donor cells in SCNT. The development of SCNT embryos show a significant difference among the four groups in blastocyst rates(16.46±0.42%, 16.13±1.26%, 17.02±1.54% VS 7.69±0.28%.) . This four fetal fibroblasts are transfected with PEGFP - C1, positive cell clones and amplified positive cell clones are compared after G418 screening. The results show that the number of positive clones and amplified cell clones of GFF1 and GFF2 are significantly higher than GFF3 and GFF4 (P < 0.05). The two strains chosen from GFF1 clones are used as donor cells, and compared by GFF1, there is no significant difference in blastocyst rates among three groups. The results show that different sources of the fetal fibroblasts also effect the development of cloned embryos ,and the efficiency of transfected exogenous gene on the developmental competence of goat SCNT embryos.
Experment 3: The present study was to investigate effects of sequential media G1/G2 and modified synthetic oviductal fluid (mSOF) culture systems on developmental competence, cell apoptosis and the pregnancy of goat somatic cell nuclear transfer (SCNT) embryos. Goat NT embryos were cultured as following: (1) 72 h in G1 then 120 h in G2.3 (group G1G2), both the culture media supplemented with 0.8% BSA (bovine serum albumin); (2) 72 h in mSOF supplemented with 0.8 % BSA then in mSOF supplemented with 10% FBS (fetal bovine serum) for 120 h (group mSOF-FBS). The results indicated that there was no significant difference between the sequential media G1/G2 and mSOF-FBS in blastocyst rates (27.7±3.1% vs 25.3±1.0%,P>0.05). Both numbers of total cell and apoptotic nuclei in blastocyst from G1/G2 were significantly higher than mSOF-FBS (109.1±6.2 vs 93.2±4.5 and 11.3±0.1% vs 4.9±0.2% respectively, P<0.05 ). However, the pregnant rates on Day 30 in group G1/G2 were higher than those in group mSOF-FBS(21.4% vs 8.0%, P<0.05 ). In conclusion, compared to mSOF-FBS, the sequential media G1/G2 could better support the development of goat somatic cell nuclear transfer embryos.
引文
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