Identification and characterization of a novel gene differentially expressed in zebrafish cross-subfamily cloned embryos.
BACKGROUND
Cross-species nuclear transfer has been shown to be a potent approach to retain the genetic viability of a certain species near extinction. However, most embryos produced by cross-species nuclear transfer were compromised because that they were unable to develop to later stages. Gene expression analysis of cross-species cloned embryos will yield new insights into the regulatory mechanisms involved in cross-species nuclear transfer and embryonic development.


RESULTS
A novel gene, K31, was identified as an up-regulated gene in fish cross-subfamily cloned embryos using SSH approach and RACE method. K31 complete cDNA sequence is 1106 base pairs (bp) in length, with a 342 bp open reading frame (ORF) encoding a putative protein of 113 amino acids (aa). Comparative analysis revealed no homologous known gene in zebrafish and other species database. K31 protein contains a putative transmembrane helix and five putative phosphorylation sites but without a signal peptide. Expression pattern analysis by real time RT-PCR and whole-mount in situ hybridization (WISH) shows that it has the characteristics of constitutively expressed gene. Sub-cellular localization assay shows that K31 protein can not penetrate the nuclei. Interestingly, over-expression of K31 gene can cause lethality in the epithelioma papulosum cyprinid (EPC) cells in cell culture, which gave hint to the inefficient reprogramming events occurred in cloned embryos.


CONCLUSIONS
Taken together, our findings indicated that K31 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos and over-expression of K31 gene can cause lethality of cultured fish cells. To our knowledge, this is the first report on the determination of novel genes involved in nucleo-cytoplasmic interaction of fish cross-subfamily cloned embryos.
Pei
De-Sheng
DS
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. peidesheng@yahoo.com.cn
Sun
Yong-Hua
YH
Chen
Chun-Hong
CH
Chen
Shang-Ping
SP
Wang
Ya-Ping
YP
Hu
Wei
W
Zhu
Zuo-Yan
ZY
D000818:Animals; D065150:Cellular Reprogramming; D019976:Cloning, Organism / Q000379:methods*; D003433:Crosses, Genetic; D004625:Embryo, Nonmammalian; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D017403:In Situ Hybridization; D053652:Nuclear Transfer Techniques; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*
D029961:Zebrafish Proteins