Generation and characterization of a stable red fluorescent transgenic Tanichthys albonubes line

Abstract

White cloud mountain minnow (Tanichthys albonubes) is a small cyprinid oviparous fresh-water fish in Southern China. For ornamental purpose, the red fluorescent protein (RFP) transgenic T. albonubes was generated by microinjection of a pMYLZ2-RFP gene construct containing zebrafish myosin light polypeptide 2 (mylz2) promoter into the fertilized eggs, and a transgenic line was established. For this line, segregation of RFP gene followed Mendelian single-gene inheritance, confirming transgene integration into the T. albonubes genome at a single locus. Results of reverse transcriptase-polymerase chain reaction (RT-PCR) analyses showed that RFP transgene was expressed in almost all body tissues, revealing an ectopic transgene expression. Based on ordinary and quantitative PCR analyses, integration pattern of RFP gene was determined to be three copies and head-to-tail multimers. An approximately 1.0 kb fragment amplified from 5'-flanking sequence of transgenes was identified in wild-type T. albonubes genome, and bears no homologous sequence in the GenBank database. In the 3'-flanking region, an approximately 1.2 kb fragment with unidentified source was amplified which has 99% homology to enterobacteria phage P1. The above results well demonstrated the pMYLZ2-RFP constructs have being inserted as a stable DNA fragment in the T. albonubes genome and could be stably inherited and expressed in transgenic progenies.

Keywords: Transgenic fish, Tanichthys albonubes, RFP, expression, flanking sequence, copy number