[Cloning of ESTs of spermatogenesis-related genes from early stage spermatogenic cells of mice using a modified DDRT-PCR method].
To avoid the shortcomings of radioactive pollution and high rate of false positives in DDRT-PCR method, the technique have been modified by replacement of radioactive reagents with fluorescent reagents followed by confirming the results using reverse RNA dot blot. The modified DDRT-PCR method was used in this study to clone spermatogenesis-related genes from early stage spermatogenic cells of mice. Primitive spermatogonia were isolated from 6-day-old mice and type B spermatogonia from 9-day-old mice. The purity of isolated cells was over 90%. Total RNA was extracted from the cells, fluorescent differential display technique was performed to screen the differentially expressed genes. Differences in expression of the screened fragments were identified by reverse RNA dot blot. 16 ESTs were selected for sequencing. The analysis results in GenBank/Blast database revealed that 7 of them were novel ESTs, and they were then registered in GenBank. All of them expressed stronger in B spermatogonia than in primitive spermatogonia. 3 of them were chosen to further identify their expression patterns by semi-quantitative RT-PCR. Compared with traditional differential display technique, the modified method used in this study can avoid radioactive pollution and eliminate false positive results. The present study suggests that the gene activation or up-regulation in B spermatogonia may be related to some specific process in the following steps of spermatogenesis.
Yu
Zuo-Ren
ZR
Department of Cell Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China.
Guo
Rui
R
Ge
Ye-Hua
YH
Guan
Ji-Kui
JK
Ma
Jing
J
Xue
She-Pu
SP
Han
Dai-Shu
DS
D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D013091:Spermatogenesis / Q000235:genetics*; D013093:Spermatogonia / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors; D015533:Transcriptional Activation
D018076:DNA, Complementary; D012313:RNA