摘要
为优化猪精子介导的基因转移(SMGT)技术,提高转基因效率,本试验利用纳米化聚酰胺-胺型树枝状聚合物(PAMAM-D)为载体介导猪精子转染外源DNA,经原位杂交检测其阳性率,体外受精(IVF)分析外源基因的表达。结果显示,0.5μg线状质粒DNA中加入PAMAM-D(质量比20∶1)阳性率最高(P<0.05);孵育时间为90、120min时,阳性率显著高于30、60min组(19.94%、18.57%与8.50%、13.87%;P<0.05);以此条件处理精子,阳性率显著高于无载体和脂质体处理组(23.93%与7.25%、13.25%;P<0.05)。各处理精子经IVF后,PAMAM-D组EGFP表达率显著高于其他2组(9.19%与4.81%、3.43%;P<0.05),各组绿色荧光胚胎经基因组PCR分析均能检测到EGFP基因。结果表明,PAMAM-D可有效介导猪精子转染外源DNA,并通过体外受精将外源基因导入胚胎中。
To optimize production of transgenic pig by sperm-mediated gene transfer, the present study was designed to examine the effects of treatment with Nano-PAMAM-D on exogenous DNA uptake of boar spermatozoa and gene expression in in vitro fertilized eggs. Exogenous DNA trea- ted with Nano-PAMAM-D was incubated with boar spermatozoa,the exogenous DNA uptake was detected by in situ hybridization, and exogenous gene expression was examined by fluorescence microscope and PCR analysis. The results showed that positive rate of sperm treated with Nano- PAMAM-D and DNA according to 0.5 vg linear plasmid plus PAMAM-D at the ratio of 20 ： 1 was the highest group （P〈0. 05）. The positive rates were significantly increased for incubation 90 or 120 min compared to incubation 30 or 60 min（19.94%,18.57% vs 8.50%,13.87%;P〈 0.05）. The complexes formed between exogenous DNA and Nano-PAMAM-D were internalized （23.93% vs 7.25%,13.25%,P〈0.05）on boar spermatozoa at a higher efficiency compared to methods using DNA or lipofectin alone. This study suggested that boar spermatozoa using Nano- PAMAM-D as transfectant could be fertilized with oocytes in vitro and that the resulting gene ofgreen fluorescent protein was detected in fertilized eggs by genomic PCR analysis. In conclusion, Nano-PAMAM-D vector could be used to efficiently introduce a exogenous gene into embryo via spermatozoa.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2013年第3期466-471,共6页
Chinese Journal of Veterinary Science
基金
上海市科委农业科技成果转化资助项目(103919N1800)
上海市科技兴农推广资助项目(2007-3-7)
上海市农科院青年科技发展基金资助项目(2011-07)
国家转基因生物新品种培育科技重大专项资助项目(2008ZX08006-005,2009ZX08006-014B)
