Integration analysis of red fluorescent protein gene(RFP) in transgenic Tanichthys albonubes.

A stable line of transgenic Tanichthys albonubes containing red fluorescent protein(RFP) gene was produced previously and RFP gene is proved transmitting in a Mendelian fashion.For further reveals the integration characteristics of RFP gene in transgenic fish,the number of construct copies,the arrangement of constructs and the nearby sequence of foreign gene inserted were detected using PCR,Southern blotting and genomic walking.The results showed that the integration pattern of exogenous gene was single locus,multi-copy(2 or 3) and head-to-tail multimers.Both fragments of 1.0 and 1.2 kb were amplified by genomic walking from 5'-and 3'-flanking sequence,respectively.Sequence analysis showed that 5'-flanking sequence existed in wild Tanichthys albonubes genome sequence,and there was no homologous sequence in the GenBank database,the 3'-flanking sequence was 99%identical to that of phage P1 partial genome,but this sequence could not be detected in wild Tanichthys albonubes genome.These result provide a evaluation basis for the genetic stability of RFP transgenic T.albonubes.