Expression of Foreign DNA Is Associated with Paternal Chromosome Degradation in Intracytoplasmic Sperm Injection-Mediated Transgenesis in the Mouse1
Expression of Foreign DNA Is Associated with Paternal Chromosome Degradation in Intracytoplasmic Sperm Injection-Mediated Transgenesis in the Mouse1
复制标题
小鼠胞浆内精子注射介导的转基因中外源 DNA 的表达与父本染色体降解相关1
作者：
M. Szczygiel;S. Moisyadi;W. Ward
摘要卵胞浆内单精子注射（ICSI）转基因技术的效率往往受到胚胎发育不良的限制。由于我们以前的工作表明，胚胎发育的损害往往与染色体异常有关，我们假设外源DNA和/或用于增强DNA整合的条件可能会诱导染色体损伤。因此，我们检查了用EGFP基因转基因产生的小鼠胚胎的染色体。精子用三种引起膜破裂的方法处理：冻融、Triton X-100或Triton X-100随后用蔗糖洗涤。将膜破裂的精子与EGFP质粒混合并注射到中期II卵母细胞中。评价了三个终点：受精卵的父系染色体、胚胎体外发育能力和转基因在桑椹胚/胚泡阶段的表达。在所有的预处理中，我们观察到一个显着的减少（约2倍）的频率正常的有核板时，精子与外源DNA孵育相比，处理时没有DNA添加。正如预测的那样，胚胎发育与受精卵的父系染色体的完整性相关。为了寻找染色体降解的可能机制，我们使用了离子螯合剂EGTA和EDTA，发现它们中和了转基因的有害影响并稳定了父本染色体。然而，在螯合剂的存在下，表达EGFP的胚胎数量与ICSI介导的转基因产生显着下降。结果表明，用外源DNA处理精子会导致受精卵中父本染色体的降解。此外，父本染色体的破坏机制和外源DNA的整合可能密切相关。
Abstract The efficiency of intracytoplasmic sperm injection (ICSI)-mediated transgenesis is often limited by poor embryo development. Because our previous work indicated that impairment of embryo development is frequently related to chromosomal abnormalities, we hypothesized that foreign DNA and/or conditions used to enhance integration of the DNA might induce chromosome damage. Therefore, we examined the chromosomes of mouse embryos produced by transgenesis with the EGFP gene. Spermatozoa were processed with three methods that cause membrane disruption: freeze-thawing, Triton X-100, or Triton X-100 followed by a sucrose wash. Membrane-disrupted spermatozoa were mixed with EGFP plasmids and injected into metaphase II oocytes. Three endpoints were evaluated: paternal chromosomes of the zygote, embryo capacity to develop in vitro, and expression of the transgene at the morula/blastocyst stage. In all pretreatments, we observed a significant decrease (approximately 2-fold) in the frequency of normal karyoplates when spermatozoa were incubated with exogenous DNA as compared with the treatment when no DNA was added. As predicted, embryo development was correlated with the integrity of the paternal chromosomes of the zygote. Searching for the possible mechanism of chromosome degradation, we used the ion chelators EGTA and EDTA and found that they neutralize the harmful effect of the transgene and stabilize the paternal chromosomes. In the presence of chelating agents, however, the number of embryos expressing EGFP produced with ICSI-mediated transgenesis decreased significantly. The results suggest that treatment of spermatozoa with exogenous DNA leads to paternal chromosome degradation in the zygote. Furthermore, the mechanisms of disruption of paternal chromosomes and the integration of foreign DNA may be closely related.
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影响因子：
4.6
作者：
Y. Kimura;Ryuzo Yanagimachi
通讯作者：
Y. Kimura;Ryuzo Yanagimachi
影响因子：
3.6
作者：
KIMURA, Y;YANAGIMACHI, R
通讯作者：
YANAGIMACHI, R
影响因子：
3.6
作者：
Kuretake, S;Kimura, Y;Yanagimachi, R
通讯作者：
Yanagimachi, R
DOI：
10.1530/jrf.0.0860679
发表时间：
1989-07-01
期刊：
JOURNAL OF REPRODUCTION AND FERTILITY
影响因子：
--
作者：
CHATOT, CL;ZIOMEK, CA;TORRES, I
通讯作者：
TORRES, I
影响因子：
56.9
作者：
GORDON, JW;RUDDLE, FH
通讯作者：
RUDDLE, FH