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1、 Transgenic Chicken with Oviduct Specific Vector AbstractThe present study was attempted to replace ovalbumin gene with tissue plasminogene activator marked with green fluorescence protein. The pl-EGFP, pl-CMV-tPAEGFP and pl-2.8OV-tPAEGFP vectors were constructed. 293FT cells were transfected with p
2、l-EGFP vector and chicken embryo fibroblasts (CEF), chicken primary germ (cPGC) cells with pl-CMV-tPAEGFP vector. The green fluorescence protein was expressed among all trasnfected cells. The pl-2.8OV-t-PAGFP and control (pEGP-N1) vector was transfected with Hela, C127 and oviduct epithelial cells.
3、The pl-2.8OV-t-PAGFP vector was only expressed in oviduct epithelial cells but not in hela and C127 while control vector was expressed in all hela, C127 and oviduct epithelial cells. The result showed that pl-EGFP, pl-CMV-tPAEGFP vectors could infect different cells but pl-2.8OV-t-PAGFP vector was o
4、nly expressed in Oviduct epithelial cells due to oviduct specific expression system. The lentivirus with (pl-2.8OV-tPAEGFP) was injected in 50 fertilized eggs, 11 (22%) chicken hatched with 4 (36%) positive containing integration of exogenous genes analyzed by DNA dot blotting and PCR. Our finding s
5、uggested that transgenic chicken can be produced using oviduct specific vector containing pl-2.8OV-tPAEGFP.Key Word: Lentiviral vector; ovalbumin gene; tissue plasminogene activator; GFP; laying hensIntroductionPresently one of the most advanced research of is genetically modified technology, it has
6、 been involved in production of transgenic animals. The rst transgenic animals were generated by infecting mouse blastocyst cells with a Moloney leukemia virus-based retroviral vector (Jaeisch 1988, 1976). Lentiviral vector has been used successfully to create transgenic mice with a very high rate o
7、f incorporation of the foreign gene (Lois et al., 2002). Lentiviral vectors allow for high levels of transgene expression in quails and chickens (Benjamine and Carlois 2006) Tissue-specic expression in transgenetic birds (PNA Sin press). Tissue plasminogen activator(tPA), a major protein involved in
8、 the breakdown of bloodclots, to treat heart attacks, strokes, clots in the lungs and is also being studied in the treatment of cancer. As anenzyme, it alsocatalyzesthe conversion ofplasminogentoplasmin, the major enzyme responsible for clot breakdown. tPA has also been used to dissolve thrombi asso
9、ciated with ischemic strokes and brain injury, people withfrostbitewho were treated with tPA had feweramputationsthan those who were not (Bruen et al 2007, Tsurupa and Medved (2001) , Ichinose et al (1986) Keeping in views the importance of tissue plasminogene activator , we have used recombinant hu
10、man tissue plasminogene to replace ovalbumin gene in magnum of oviduct of laying hens. Ovalbumin is the major protein of the magnum of the hens oviduct and is not normally found in other tissues (Kohler et al 1968）. The chicken ovalbumin gene encodes more than half of egg white protein， with approxi
11、mately 2.2 g per egg (Burley and Vadehra, 1989). The oviduct of laying hen has approximately 105 copies of the ovalbumin mRNA per cell (Palmiter, 1975). Ovalbumin constitutes 54% of the protein in the egg white and its steroid-responsive tissue-specific regulation is well documented (Dougherty and S
12、anders, 2005). We constructed pl-2.8OV-T_PAGFP vectors by cloning oviduct specific promoter from ovalbumin gene of laying hens and human tissue plasmingene activator.Materials and Method增强型绿色荧光蛋白基因（ EGFP ）来自 pEGFP 质粒 (GeneBank Accession No: U55762 本室保存 ) ，由 pEGFP 质粒经 Nde 、 Xba （ NEB 公司）双酶切后经琼脂糖凝胶电泳分
13、离纯化后获取pEGFP plasmid (already saved in our laboratory and the pL-lacZ plasmid (Invitrogen) containing (lentiviral vector sequence) were digested by Nde 1, Xba 1 enzymes and the new plasmid named pL-EGFP . The newly constructed plasmid was载体去磷酸化及快速连接方法参见相关产品附带说明。 purified by相同的酶酶切 pL-lacZ 质粒（ Invitrog
14、en ）获取含慢病毒转运载体序列的 DNA 片段，以热酶磷酸酶进行载体去磷酸化（ NEB ），琼脂糖凝胶电泳分离纯化后以快速连接试剂盒（ LigaFastTM Rapid DNA Ligation System, Promega ）与 EGFP 连结，最后转染 JM109 感受态细菌扩增质粒，新质粒命名为 pL-EGFP （图 1-1 ）。 Quick Connect Kit (Liga Fast TM Rapid DNA Ligation System, Promega).Fig 1-1 The construction of pL-EGFP plasmid The tissue plasm
15、inogen activator gene with pGEM-tPA plasmid was received from Professor Tao Xi, China Pharmaceutical University. For the insertion of tissue plasminogene activator into the pL-EGFP vector, t-PA primers were designed with sense primer 5GCGGGAATTCTCGAGATGGATGCAA3 and Antisense Primer 5GATTATCACGGATCGA
16、TGTTGTCAC3 with Xho1 and BamH1 restriction site. The 20L PCR reaction system to include: 50ng genomic DNA, 2l PCR buffer, 0.2 mM dNTPs, 10M primer, 0.5 units of Taq DNA polymerase (Promega).PCR reaction conditions : 94 denaturation 2 min; 94 30s, 58 30 s and 72 2 min total of 32 cycles, final extension 72 for 7 min. The
