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首页> 外文期刊>African Journal of Biotechnology >Effect of potassium simplex optimization medium (KSOM) and embryo screening on the production of human lactoferrin transgenic cloned dairy goats
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Effect of potassium simplex optimization medium (KSOM) and embryo screening on the production of human lactoferrin transgenic cloned dairy goats

机译：单纯钾优化培养基（KSOM）和胚胎筛选对人乳铁蛋白转基因克隆奶山羊生产的影响

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摘要

In this study, we produced cloned transgenic dairy goat based on dairy goat ear skin fibroblast as donor cells for nuclear transfer (NT), which were modified by human lactoferrin (hLF) gene. The developmental competence of NT embryos was compared with either between different embryo culture medium, potassium simplex optimization medium (KSOM) and tissue?culture medium (TCM 199), or different classification of NT embryos (48 h after fusion). First we cultured NT embryos to cleavage stage (48 h after fusion) by TCM 199 supplemented with 1 mg/ml bovine serum albumin BSA and KSOM, then used TCM 199 supplemented with 10% FBS to culture them to blastula stage. The results show that the NT embryos in KSOM (19.5%) were superior to TCM 199 (10.6%) in blastulation. In the second experiment, we found that the growth rate of NT embryos (48 h after fusion) was different, then we divided them into four groups: 2-cell, 3- to 4-cell, 5- to 8-cell and >8-cell in stereo microscope and cultured them in vitro respectively. The results show day-2 embryos at 3-4cell and 5-8cell stage (31.9 and 28.2%, P 8-cell (8.3%) stage, and finally three healthy cloned transgenic goat were successfully produced using 3-8 cell embryos at Day-2 (82%). Using Hoechst 33342 staining, we also found that the >8 cells embryos at Day-2 demonstrated higher frequency of fragmentation, which may be the one cause of the low blastocyst formation rate. This study therefore demonstrates that KSOM medium could be selected as the early embryo culture medium, and 3-8 cell embryos at day-2 (48 h after fusion) may be the suitable embryos for transplantation, which could reduce the nuclei fragmentation and result in good quality blastocysts that may also enhance the efficiency of transgenic cloned dairy goats production, as well as decrease the economic loss due to embryonic mortality when embryos are transferred to synchronized recipients.
机译：在这项研究中，我们生产了基于乳山羊耳朵皮肤成纤维细胞的克隆转基因乳山羊作为供体细胞，用于核转移（NT），并被人乳铁蛋白（hLF）基因修饰。将NT胚胎的发育能力与不同的胚胎培养基，单纯钾优化培养基（KSOM）和组织培养基（TCM 199）或NT胚胎的不同分类（融合后48小时）进行比较。首先我们通过添加1 mg / ml牛血清白蛋白BSA和KSOM的TCM 199将NT胚胎培养至分裂期（融合后48小时），然后使用添加10％FBS的TCM 199将其培养至囊胚期。结果表明，KSOM中的NT胚（19.5％）在中胚层形成方面优于TCM 199（10.6％）。在第二个实验中，我们发现NT胚胎（融合后48小时）的生长速率不同，然后将它们分为四组：2细胞，3至4细胞，5至8细胞和>立体显微镜中的8细胞，分别在体外培养。结果显示，第2天的胚胎处于3-4个细胞和5-8个细胞的阶段（分别为31.9和28.2％，P 8个细胞（8.3％）），最后在第3天使用3-8个细胞胚胎成功地生产了三只健康的克隆转基因山羊-2（82％）。使用Hoechst 33342染色，我们还发现，第2天> 8个细胞的胚胎表现出更高的碎片化频率，这可能是胚泡形成率低的原因之一，因此该研究表明KSOM可以选择该培养基作为早期胚胎培养基，并且第2天（融合后48小时）的3-8个细胞胚胎可能是适合移植的胚胎，可以减少细胞核破裂并产生高质量的胚泡，这可能也可以增强了转基因克隆奶山羊生产的效率，并减少了将胚胎转移到同步受体时因胚胎死亡而造成的经济损失。

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