Vichera G, Moro L N, Buemo C, Salamone D
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, Capital Federal (C1417), Argentina.
Zygote. 2014 May;22(2):195-203. doi: 10.1017/S0967199412000433. Epub 2012 Oct 1.
Summary This study was designed to evaluate the quality and viability of bovine embryos produced by in vitro fertilization (IVF), after intracytoplasmic injection of pCX-EGFP-liposome complexes or pBCKIP2.8-liposome complexes (plasmids that codify the human insulin gene). Cleavage, blastocysts and expanded blastocysts rates of these both groups were not different from that of controls (IVF or IVF embryos injected with liposomes alone; IVF-L). The percentage of EGFP-positive (EGFP+) blastocysts was 41.8%. In Experiment 2, the blastocysts obtained after injection of pCX-EGFP-liposome complexes that did or did not express the transgene, were analyzed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) assay at days 6, 7 and 8 of culture in vitro(Bd6, Bd7 and Bd8), in order to evaluate DNA fragmentation. The EGFP+ blastocysts showed different proportions of TUNEL-positive cells (T+) at Bd6, Bd7 and Bd8 (91, 73.7 and 99.5%, respectively) while blastocysts without EGFP expression (EGFP-) showed statistically lower numbers of fragmented nuclei (0, 44.6 and 85%, respectively; P < 0.05). There was no evidence of DNA fragmentation in either Bd6 or Bd7 IVF and IVF-L control blastocysts, but T+ nuclei were detected at Bd8 in both groups (66.4 and 85.8% respectively). Finally, IVF blastocysts (n = 21) injected with insulin-liposome complexes, cultured for 6, 7 and 8 days, were transferred to recipient cows. Pregnancy rates of 18.2% (2/11) and 40% (2/5) resulted from the transfer of Bd6 and Bd7 cells, respectively. Two pregnancies developed to term but they were not transgenic for the insulin gene. In conclusion, EGFP expression affects DNA integrity but not embryo development. Moreover, additional transfers are required in order to overcome the drawbacks generated by in vitro culture length and transgene expression.
摘要 本研究旨在评估体外受精（IVF）产生的牛胚胎在胞质内注射pCX-EGFP-脂质体复合物或pBCKIP2.8-脂质体复合物（编码人胰岛素基因的质粒）后的质量和活力。这两组的卵裂率、囊胚率和扩张囊胚率与对照组（IVF或仅注射脂质体的IVF胚胎；IVF-L）无差异。EGFP阳性（EGFP+）囊胚的百分比为41.8%。在实验2中，对注射了表达或未表达转基因的pCX-EGFP-脂质体复合物后获得的囊胚，在体外培养的第6、7和8天（Bd6、Bd7和Bd8）通过TUNEL（末端脱氧核苷酸转移酶dUTP缺口末端标记）分析来评估DNA片段化。EGFP+囊胚在Bd6、Bd7和Bd8显示出不同比例的TUNEL阳性细胞（T+）（分别为91%、73.7%和99.5%），而无EGFP表达的囊胚（EGFP-）显示出统计学上较低的核片段化数量（分别为0、44.6%和85%；P<0.05）。在Bd6或Bd7的IVF和IVF-L对照囊胚中均未发现DNA片段化的证据，但两组在Bd8均检测到T+核（分别为66.4%和85.8%）。最后，将注射了胰岛素-脂质体复合物并培养6、7和8天的IVF囊胚（n = 21）移植到受体母牛体内。Bd6和Bd7细胞移植后的妊娠率分别为18.2%（2/11）和40%（2/5）。有两次妊娠足月，但它们并非胰岛素基因转基因。总之，EGFP表达影响DNA完整性，但不影响胚胎发育。此外，为克服体外培养时间和转基因表达产生的缺点，需要进行额外的移植。
