Foreign DNA introduced into the vas deferens is gained by mammalian spermatozoa
Foreign DNA introduced into the vas deferens is gained by mammalian spermatozoa
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哺乳动物精子获得引入输精管的外源DNA
DOI：
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发表时间：
1998
影响因子：
2.5
通讯作者：
P. Esponda
中科院分区：
文献类型：
--
作者：
E. Huguet;P. Esponda
利用体外和体内方法分析了小鼠和大鼠精子对外源DNA的摄取。使用两种DNA构建体，一种含有生长激素（GH）基因，另一种含有与αA-晶状体蛋白启动子（CPV-1质粒）连接的c-myc癌基因。对于体外方法，将洗涤的附睾精子在线性化DNA存在下孵育2小时。对于体内实验，将DNA注射到输精管的近端区域，6小时后回收精子。采用荧光标记原位杂交和电镜技术对外源基因在精子中的定位。利用激光共聚焦显微镜进行三维重建，可以观察到外源DNA在精子中的精确定位。在体外或体内处理后，60-70%的精子发生了外源DNA的摄取。在精子核中检测到阳性信号，并且不受DNA酶处理的影响。利用从精子核中分离的DNA和相应的标记探针，通过狭缝印迹和PCR技术评估外源DNA的掺入。从体内处理的精子中分离的DNA与CPV-1质粒之间的核苷酸序列比较显示98.6%的同一性。这些结果显示了精子在体内掺入外源DNA的能力，这种DNA到达细胞核的能力，并且还表明附睾和输精管分泌物不阻断这些能力。摩尔Reprod. Dev. 51：42-52，1998.© 1998 Wiley利斯公司
The uptake of exogenous DNA by mouse and rat spermatozoa was analyzed using in vitro and in vivo methods. Two DNA constructs were used, one containing the Growth hormone (GH) gene and the other the c‐myc oncogene linked to the αA‐crystallin promoter (CPV‐1 plasmid). For the in vitro approach, washed epididymal spermatozoa were incubated for 2 hr in the presence of linearized DNA. For in vivo experiments, DNA was injected into the proximal region of the vas deferens, and spermatozoa were recovered 6 hr later. In situ hybridization employing fluorescent markers and electron microscopy were used to localize the exogenous genes in spermatozoa. The precise localization of the foreign DNA in spermatozoa was visualized by tridimensional reconstructions using a confocal laser microscopy. Uptake of exogenous DNA occurred in 60–70% of the spermatozoa after in vitro or in vivo treatments. A positive signal was detected in the sperm nucleus and was not affected by DNase treatments. Incorporation of exogenous DNA was also evaluated by slot blot and PCR techniques using the DNA isolated from the sperm nuclei and the corresponding labelled probes. Comparison of a nucleotide sequence between the DNA isolated from in vivo treated spermatozoa and CPV‐1 plasmid showed a 98.6% identity. These results show the in vivo capacity of spermatozoa to incorporate exogenous DNA, the ability of this DNA to reach the nucleus, and also demonstrate that epididymal and vas deferens secretions do not block these capacities. Mol. Reprod. Dev. 51:42–52, 1998. © 1998 Wiley‐Liss, Inc.
DOI：
10.1073/pnas.91.24.11303
发表时间：
1994-11-22
影响因子：
11.1
作者：
BRINSTER, RL;AVARBOCK, MR
通讯作者：
AVARBOCK, MR