Transgenic quail production by microinjection of lentiviral vector into the early embryo blood vessels.

Zhiyi Wan,Peng Sun,Fuxian Yu,Tao Wang,Xiuhcun Tian,Zandong Li,Qiang Shao,Zifu Zhang,Li Yan,Fang Yuan,Wenxin Zhang

doi:10.1371/journal.pone.0050817

Abstract

Several strategies have been used to generate transgenic birds. The most successful method so far has been the injection of lentiviral vectors into the subgerminal cavity of a newly laid egg. We report here a new, easy and effective way to produce transgenic quails through direct injection of a lentiviral vector, containing an enhanced-green fluorescent protein (eGFP) transgene, into the blood vessels of quail embryos at Hamburger-Hamilton stage 13–15 (HH13–15). A total of 80 embryos were injected and 48 G0 chimeras (60%) were hatched. Most injected embryo organs and tissues of hatched quails were positive for eGFP. In five out of 21 mature G0 male quails, the semen was eGFP-positive, as detected by polymerase chain reaction (PCR), indicating transgenic germ line chimeras. Testcross and genetic analyses revealed that the G0 quail produced transgenic G1 offspring; of 46 G1 hatchlings, 6 were transgenic (6/46, 13.0%). We also compared this new method with the conventional transgenesis using stage X subgerminal cavity injection. Total 240 quail embryos were injected by subgerminal cavity injection, of which 34 (14.1%) were hatched, significantly lower than the new method. From these hatched quails semen samples were collected from 19 sexually matured males and tested for the transgene by PCR. The transgene was present in three G0 male quails and only 4/236 G1 offspring (1.7%) were transgenic. In conclusion, we developed a novel bird transgenic method by injection of lentiviral vector into embryonic blood vessel at HH 13–15 stage, which result in significant higher transgenic efficiency than the conventional subgerminal cavity injection.

Highlights

The study of birds makes important contributions to scientific knowledge
As an improvement to the previous approaches, we have developed a novel method incorporating the injection of a lentiviral vector directly into the blood vessels of Hamburger-Hamilton stage 13–15 (HH13–15) quail embryos which avoids the manipulation of primordial germ cell (PGC) in vitro
Transgene detection in chimeric quail Quail embryos at stage HH13–15 were chosen to injecte the lentiviral vector containing the enhanced-green fluorescent protein (eGFP) gene driven by a cytomegalovirus (CMV) promoter, because the blood vessels are too fine to Injection methods Subgerminal cavity injection Blood vesse linjection P value

Summary

Introduction

The study of birds makes important contributions to scientific knowledge. Birds are used as animal models in biological research and the avian oviduct can serve as a bioreactor for the production of valuable proteins in the egg [1]. Lyall et al have successfully generated transgenic chickens that were resistant to avian influenza [11] The infection of these blastoderm cells in subgerminal cavity by lentivirus successfully produced G0 transgenic chimeras and transgenic G1 birds, but random integration of the transgene frequently resulted in transcriptional blockade [4]. As an improvement to the previous approaches, we have developed a novel method incorporating the injection of a lentiviral vector directly into the blood vessels of HH13–15 quail embryos which avoids the manipulation of PGCs in vitro. This method generates transgenic germ line chimeras, that are able to produce transgenic offsprings. This new approach should make it easier to manipulate the avian genome and may lead to novel biotechnological applications

Materials and Methods

Results

Discussion