Effects of G418 treatment of donor cells on the in vitro developmental efficiency of cloned porcine embryos
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摘要:目的
探索G418处理供体细胞对其核移植胚胎发育效率的影响。
方法利用G418单独处理猪成体成纤维细胞6 d后，收集细胞，利用荧光定量PCR的方法检测处理前后细胞中抗氧化应激、细胞凋亡相关基因的表达水平，运用亚硫酸盐结合测序法分别检测基因组重复序列LINE-1、微卫星的DNA甲基化状态，以及其核移植胚胎体外发育的能力。
结果经不同质量浓度G418处理的供体细胞的抗氧化应激酶相关基因及细胞凋亡基因表达发生显著变化(P＜0.05)，但其DNA甲基化水平没有发生改变(P＞0.05)；经G418处理的供体细胞的核移植胚胎的体外发育效率显著低于对照组(P＜0.05)。
结论G418处理供体细胞可能对其克隆胚胎体外发育效率有抑制作用。
Abstract:ObjectiveTo explore the influence of G418 treatment of donor cells on the developmental efficiency of somatic cell nuclear transfer (SCNT) embryos.
MethodPorcine adult fibroblasts were collected after six days of G418 treatment. Expression levels of antioxidation and apoptosis-related genes were detected by quantitative RT-PCR before and after G418 treatment. The DNA methylation levels of LINE-1 repetitive sequences and microsatellites were analyzed using bisulfites sequencing analysis, and the in vitro developmental rates of SCNT embryos were investigated.
ResultG418 treatment of donor cells caused significant (P < 0.05) changes in expression levels of genes related to antioxidation and apoptosis, but did not significantly(P > 0.05) change DNA methylation levels. SCNT embryos cloned from G418-treated donor cells exhibited a significantly (P < 0.05) lower in vitro developmental rates compared with the control group.
ConclusionG418 treatment of donor cell may inhibit the development of SCNT embryo.
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Keywords:
- G418 /
- porcine /
- cloning /
- nuclear transfer /
- transgene /
- embryonic development
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图 1 G418筛选前后的猪成体成纤维细胞
A、B分别为G418筛选前、后的猪成体成纤维细胞(100×)。
Figure 1. Porcine adult fibroblasts after G418 screening
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图 2 不同质量浓度G418处理对猪成体成纤维细胞抗氧化酶相关基因表达的影响
图中同一基因不同处理间柱上凡是有一个相同小写字母者，表示差异不显著(P > 0.05, χ2检验)。
Figure 2. Relative expression of antioxidation-related genes in porcine adult fibroblasts treated with different concentrations of G418
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图 3 不同质量浓度G418处理对猪成体成纤维细胞凋亡相关基因表达的影响
图中同一基因不同处理间柱上凡是有一个相同小写字母者，表示差异不显著(P > 0.05, χ2检验)。
Figure 3. Relative expression of apoptosis-related genes in porcine adult fibroblasts treated with different concentrations of G418
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图 4 不同质量浓度G418处理猪成体成纤维细胞基因组重复序列甲基化水平的影响
图中1~3为3个处理组个体重复；1条链表示1个克隆测序结果，1个圈表示1个CpG，其中，黑色实心圆圈表示甲基化的CpG，白色空心圆圈表示未甲基化的CpG，串珠图中“ǀ ”表示该位点发生非CG或TG突变。A、B、C分别为LINE-1对照组0 μg·mL-1G418、100 μg·mL-1 G418处理组、200 μg·mL-1 G418处理组; D、E、F分别为微卫星对照组0 μg·mL-1G418、100 μg·mL-1 G418处理组、200 μg·mL-1 G418处理组。
Figure 4. DNA methylation levels of genomic repeat sequences in porcine adult fibroblasts treated with different concentrations of G418
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图 5 不同质量浓度G418处理对猪成体成纤维细胞DNA甲基化酶相关基因表达的影响
图中同一基因不同处理间柱上凡是有一个相同小写字母者，表示差异不显著(P > 0.05, χ2检验)。
Figure 5. Relative expression of DNA methylation related genes in porcine adult fibroblasts treated different concentrations of G418
表 2 G418处理猪成体成纤维细胞对猪体细胞克隆胚胎体外发育性能的影响1)
Table 2 The effects of G418 on the in vitro development of porcine somatic cell nuclear transfer embryos
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