【作者】 燕海峰；

【导师】 柳小春；

【作者基本信息】 湖南农业大学 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 为建立家鸡的高效基因转移技术,本文从种蛋开窗技术的改进、用慢病毒表达载体将绿色荧光蛋白(GFP)基因转染种蛋内囊胚细胞和原生殖细胞、逆转录病毒载体转染睾丸细胞、人促红细胞生成素(hEPO)基因家鸡输卵管组织特异性表达载体的构建等方面进行了研究,结果如下:1改进了种蛋开窗工具、开窗方法以及封口方法,使该方法更简单实用,制作效率提高了2倍以上,便于在实践中推广应用,并已申报国家发明专利(申请号CN200710034253.0,公开号CN101020898)。为解决目前因外源基因的随机整合,需要大量实验样本才能使转基因家鸡研究获得成功等问题奠定了基础。2家鸡种蛋自身含有禽胚胎干细胞(BCs)或原生殖细胞(PGCs)。采用Gateway技术构建了含绿色荧光蛋白(GFP)基因的pLenti6/v5-DEST-GFP慢病毒表达载体,转染体外培养的BCs,获得了约70%的表达效率。转染体内囊胚,孵化13d时,PCR检测阳性率为64.7%。转染血液循环中的PGCs:孵化率为35.0%,在出壳后死亡的3只小鸡肝脏中,GFP基因检出率为100%,其中一只小鸡的眼部能检测到绿色荧光;存活的4只鸡中有3只在12月龄的血液样品中,经PCR检测扩增出了GFP基因片段。3睾丸生精上皮内含有二倍体的精原干细胞,将这些细胞在体外转染,然后移植入经不育处理的受体公鸡睾丸,使其恢复生精能力并产生转基因精子,是家鸡基因转移的一种新方法。本文构建了含GFP基因的pLG逆转录病毒载体,滴度达到5×10~7 IU/mL,转染了体外培养的睾丸细胞,转染后培养到5 d时,流式细胞仪检测GFP基因的表达率为37.0%,碘化丙锭(PI)染色检测出的细胞活力达99.7%,而且整合的前病毒CpG甲基化程度相当低。转染后的睾丸细胞移植到7只经不育处理的公鸡睾丸,其中2只在2-3个月恢复了生精能力,经PCR扩增证实其产生的精子中含有转基因。4外源基因仅在输卵管组织中特异表达是家鸡转基因技术所追求的目标。从鸡输卵管基因组DNA中扩增出1.3kb的OV(卵清蛋白)基因5’端调控区,作为家鸡输卵管组织特异性表达启动子。从phEBS-HB质粒扩增出了2.1kb的hEPO(人促红细胞生成素)基因全长DNA片断,作为目的基因。为方便EPO的检测,构建了含hEPO和GFP的共表达载体pOV-GFP-hEPO。通过测序和转染体外培养的家鸡输卵管上皮细胞,证实载体构建正确并能表达。更多还原

【Abstract】 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.更多还原