Analysis of transgenic quail produced by pantropic retroviral vector

Abstract

The present study was conducted to produce the transgenic quail by using pantropic retroviral vector containing human growth hormone (hGH) gene and to increase the efficiency of transgenesis through several technical approaches. The constructed retroviral vector in this study included human growth hormone gene as a functional gene and neomycin resistance gene for G418 selection during in vitro cell culture. Specially, pantropic retroviral gene transfer system, which is mediated by vesicular stomatitis virus G protein (VSV-G), was used for producing the transgenic quail. For the expression and titering of hGH-retroviral vector, the concentration of hGH were compared in quail embryonic fibroblast and NIH3T3 cells by ELISA. The hGH was produced 8.63ng/ml and 116.1ng/ml in quail embryonic fibrobalsts without and with G418 selection of packing cell lines, respectively. The retrovirus then was injected into blood vessel of 2-day-old quail embryos and incubated until further next experiments. The hatchability was 26.0% and 28.6% in the non-concentrated and concentrated retrovirus injection, respectively. For screening of foreign transgene, polymerase chain reaction (PCR) and Southern blotting was conducted at 6, 12-days, and hatch after transfer of retrovirus. As a result of PCR and Southern blotting, hGH gene was detected in several embryonic tissues including the embryonic gonads, regardless of the embryonic developmental stages. Of total 29 analyzed quails, 19 quails were positive to hGH gene sequence with 65.5% of transgenic efficiency. Thus, transgenic aves can be efficiently produced by using pantropic retroviral vector and increase the germline transmission of transgene by the microinjection of highly concentrated retroviral vector into blood vessel.