Green A L, Wells D N, Oback B
Ruakura Research Centre, Reproductive Technologies, AgResearch Ltd., Hamilton, New Zealand.
Biol Reprod. 2007 Sep;77(3):395-406. doi: 10.1095/biolreprod.106.058164. Epub 2007 May 23.
It has been postulated that mammalian nuclear transfer (NT) cloning efficiency is inversely correlated with donor cell differentiation status. To test this hypothesis, we compared genetically identical and increasingly differentiated donors within the myogenic lineage. Bovine male fetal muscle cells were cultured for 1-6 days in vitro. The proportion of cells displaying the following antigens was quantified by immunofluorescence microscopy: MYOD1, MYF5, PAX7, MYOG, DES, MYH, and 5-Bromo-2-deoxyuridine. Based on the antigen profile of both bulk populations and individually size-selected cells prepared for NT, donors serum-starved for 1, 4, and 5 days were classified as myogenic precursors (MPCs), myotubes (MTs), and muscle-derived fibroblasts (MFs) with purities of 92%, 85%, and 99%, respectively. Expression of the following transcripts was measured by RT-PCR in 1) cells selected for NT, 2) metaphase II oocytes, 3) NT couplets, 4) NT reconstructs, 5) NT two-cell embryos, and 6) NT blastocysts: MYOD1, MYF5, PAX7, MYOG, MYF6, ACTB, and 18S rRNA. Muscle-specific genes were silenced and remained undetectable up to the blastocyst stage, whereas housekeeping genes 18S and ACTB continued to be expressed. Differentiation status affected development to transferable embryos (118 [23%] of 520 vs. 93 [11%] of 873 vs. 66 [38%] of 174 for MPC vs. MT vs. MF, respectively, P < 0.001). However, there were no significant differences in pregnancy rate and development to weaning between the cell types (pregnancy rate: 14 [64%] of 22 vs. 8 [35%] of 23 vs. 10 [45%] of 22, and development: 4 [18%] of 22 vs. 2 [9%] of 23 vs. 3 [14%] of 22 for MPC vs. MT vs. MF, respectively).
据推测，哺乳动物核移植（NT）克隆效率与供体细胞分化状态呈负相关。为验证这一假设，我们比较了肌源性谱系内基因相同且分化程度逐渐增加的供体细胞。将牛雄性胎儿肌肉细胞在体外培养1 - 6天。通过免疫荧光显微镜对显示以下抗原的细胞比例进行定量：MYOD1、MYF5、PAX7、MYOG、DES、MYH和5 - 溴 - 2 - 脱氧尿苷。根据用于核移植的总体细胞群以及单独按大小分选的细胞的抗原谱，将饥饿培养1天、4天和5天的供体细胞分别归类为肌源性前体细胞（MPC）、肌管（MT）和肌肉来源的成纤维细胞（MF），纯度分别为92%、85%和99%。通过逆转录 - 聚合酶链反应（RT - PCR）检测以下转录本在以下细胞中的表达：1）用于核移植的细胞、2）中期II卵母细胞、3）核移植联体、4）核移植重构胚、5）核移植二细胞胚胎和6）核移植囊胚：MYOD1、MYF5、PAX7、MYOG、MYF6、ACTB和18S rRNA。肌肉特异性基因被沉默，直至囊胚阶段仍未检测到，而管家基因18S和ACTB持续表达。分化状态影响可移植胚胎的发育（MPC、MT和MF分别为520个中的118个[23%]、873个中的93个[11%]和174个中的66个[38%]，P < 0.001）。然而，不同细胞类型之间的妊娠率和断奶发育情况没有显著差异（妊娠率：MPC、MT和MF分别为22个中的14个[64%]、23个中的8个[35%]和22个中的10个[45%]；发育情况：MPC、MT和MF分别为22个中的4个[18%]、23个中的2个[9%]和22个中的3个[14%]）。
