Studies On Transgenic Chicken Using Pluripotent Stem Cells Transfected By Retrovirus

Firstly we developed the egg windowing techniques. At the same time we studied the system to make transgenic chicken using the BCs, PGCs and TCs as target cells, the retroviral and lentiviral vector with GFP as non oviduct-tissue-specific expressing vector. Secondly we cloned the 5' flanklying fragment of the OV gene and the hEPO gene so as to construct the oviduct-tissue-specific plasmid for producing the human protein using chicken. The following were the results:1 We invented a technique for windowing the fertile eggs, which the cover cut from the eggshell could recover automatically on the window. Based on this technique several factors affecting the hatchability of the windowed eggs (HWE) were investigated, such as different seal method, different windowing spot on the eggshell and adding liquid to drive air bubbles within the windowed eggs. It was indicated that the seal methods here did not remarkably affect the HWE and the highest results got up to 65%; It seemed as if that adding liquid to move the air bubbles did not help to increase the HWE, and the volume of the bladder has also no visible effect on the HWE; The HWE of the method, which windowed on the blunt end of the eggs (access to the embryo by stinging through the inner membrane) was much higher than that of windowed on equatorial plane, and the rate of both methods to find the embryos was 90%; The efficiency was 30 eggs for one person one hour using the above methods to window, find embryo and recover the eggs. The technique reported here was very efficiency and practical, and it would do great helps to the research on transgenic poultry as well as relative fields.2 To develop a method for transgenic chicken mediate by lenfiviral vector. We amplified the attBl-EGFP-attB2 sequence by PCR, and constructed pLenti6 / v5-DEST-EGFP vector by Gateway technology. Four Lentiviral vectors co-transfected of the packing cell line mediated with the calcium phosphate. The lenfiviral particle was collected and purified, and the titer was determined by transfection of the 293FT cell line. We confirmed that the construction was right by sequencing and the titer was 2×109 TU/mL. The rate of transfection was about 70% when observed under the fluorescence microscope. The fertile eggs at the stage of BCs and PGCs were windowed, and the Lentivrial vector was microinjected into the eggs. For BCs fertile eggs, the positive embryos was 64.7% (11/17) when the 13-day embryos were checked by PCR. The results of the experiment of the PGCs stage fertile eggs (experiment group 1): the hatchability was 35%; there were no positive embryos checked by PCR among the early died ones; Three chickens died little time after hatching, and they were all positive when checked using the liver DNA by PCR, among which there was one chicken emitting green light at the part of the eye; At the age of 1 month and 6 month, there were three chickens among four alive ones (75%) was positive when checked the blood DNA by PCR, however they were very weak positive at the age of 12 month.3 In this study, we present a successful retroviral infection of chicken testicular cells and consequent transduction of the retroviral vector into the sperm of recipient cockerels. A vesicular stomatitis virus glycoprotein G-pseudotyped recombinant retroviral vector, carrying the enhanced green fluorescent protein reporter gene was applied to the short-term culture of dispersed testicular cells. The efficiency of infection and the viability of infected cells were analyzed by flow cytometry. No significant CpG methylation was detected in the infected testicular cells, suggesting that epigenetic silencing events do not play a role at this stage of germ line development. After transplantation into sterilized recipient cockerels, these retrovirus-infected testicular cells restored exogenous spermatogenesis within 9 weeks with approximately the same efficiency as non-infected cells. Transduction of the reporter gene encoding the green fluorescent protein was detected in the sperms of recipient cockerels with restored spermatogenesis. Our data demonstrate that, similarly as in mouse and rat, the transplantation of retrovirus-infected spermatogonia provides an efficient system to introduce genes into the chicken male germ line.4 The 1.3kb chicken OV gene 5'-flanking reguratory region was amplified from the genome of chicken oviduct tissue and hEPO gene was amplified from the plasmid phEBS-HB. Then the hEPO was cloned into the MCS of pEGFP-C1(named pEGFP-hEPO) and then the chicken ovalbumin 5'- flanking reguratory region was cloned into pEGFP-hEPO which was digested by Ase I and Vsp I (named pOV-GFP-hEPO) in order to replace the CMV promoter. The plasmid was confirmed by sequenced and restriction endonuclease reaction. Then the GFP expression construct was transfected into the primary chicken oviduct epithelial cells by Lipofectin and detected green fluorescence. It was demonstrated that the pOV-GFP-hEPO vector can express in the special location of chicken oviduct epithelial cells.