| The manipulation among chicken genome has offered powerful bioreactor for production of pharmaceutical and industrial proteins. The laying hen is major candidate of bioreactor for producing pharmaceutical protein due to expression of specific characters in egg. Chicken ovalbumin promoter is the major strongest tissue-specific promoter that yields more than 50% of the total egg white of egg. In the frist part of this study, we constructed oviduct specific vector (pL-2.8OVtPAGFP) containing tissue plasminogen activator protein (tPA) coupled by GFP and checked its in vitro, in vivo expression in laying hen and in fertilized eggs. For in vitro analysis, oviduct epithelial cells, HeLa, C127 and 293FT cells were cultured and transfected with pL-2.8OVtPAGFP and pEGP-N1 (control) vector, respectively. The pL-2.8OVtPAGFP vector was only expressed in oviduct epithelial cells but not in HeLa, C127 and 293FT cells whereas pEGP-N1 vector was expressed in oviduct epithelial, HeLa, C127 and 293FT cells. For in vivo analysis, the pL-2.8OVtPAGFP vector was injected in laying hens through wing vein and their eggs, tissues were observed for (tPAGFP) expression. The western blot of treatment group eggs and oviduct epithelial cells protein denoted 89kDa band which reveal that tPAGFP has been expressed in egg white and oviduct epithelial cells. The fibrinolysis of tPA in egg white, oviduct cells protein showed dark zone around samples, that confirmed tPA activity. The GFP was observed in only oviduct tissues but not in heart, muscles, liver and intestinal tissues of vector injected hens. The lentivirus containing (pL-2.8OVtPAGFP) was produced, injected into 50 fertilized eggs and hatched chicks were assayed for (tPAGFP) expression. It was observed that 11 chicks hatched and only four (36%) of (11), including two male and two female were found carrier of (tPAGFP) by PCR analyses. The Go generation cockrele was crossed with wild type hens and eggs were collected for G1 generation. It was found that only 4 chicks in G1 generation were detected positive containing the (tPAGFP) The GFP in hatched chicks was seen in only oviduct tissues but not in testes and other tissues of the hen and cockrele. The present study is the first successful evident in reporting in vitro and in vivo expression of (tPAGFP) in oviduct epithelial cells, egg white and tissues of vector injected hens. It was also revealed that injection of lentivirus with (pL-2.8OVtPAGFP) in fertilized eggs, human recombinant tPA is expressed in G0 and G1 generation of chick. It is concluded that using further research on oviduct specific vector, relatively high and authentic tPA can be produced in laying hens.In the second part of study, oviduct specific expression of human recombinant leptin was observed in laying hens. The oviduct specific vector containing leptin (pL-OV-Leptin) was successfully constructed and the lentivirus containing (pL-OV-Leptin) was produced and injected in fertilized eggs. The 7 chicks hatched in Go generation. The PCR analysis revealed that all hatched chicks were detected carrier of human recombinant leptin. The Go generation birds are being grown and will be mated with wild type hens to get further generation for confirming transgen silencing in F1 and F2 generations and assay level of leptin in their eggs. It is frist successful evident in reporting the human recombinant leptin in laying hens.In the third part of study, the effect of first intron on ovalbumin promoter activity was investigated using immortalization of oviduct epithelial cells. The oviduct epithelial cells were cultured and then immortalized using pL-CTAGNEO virus. The pL-CTAGNEO virus maintained the structural integrity and growth of oviduct epithelial cells for 18 days whereas growth rate declined continuously with aging and cellular death after 7-10 days in controlled culture. The immortalized oviduct epithelial cells were transfected with pL-OV1345tPAGFP and pL-OV2964tPAGFP virus, respectively. The DNA and mRNA abundance of OV1345 virus was 46 and 14 times higher than OV2964 virus. The transcript effect of OV2964 virus was 3-fold higher than OV1345. The result showed that chicken oviduct epithelial cell line can be established using pL-CTAGNEO virus and used as continous and stable in vitro system for studing the oviduct epithelial cells. It was also observed that first intron in ovalbumin gene has a cis-regulation function for transcription.In the fourth part of study, we confirmed new method of drilling, sealing eggs and observed the hatchablity of five different groups. The 250 fertilized eggs were divided into A, B, C, D and E groups, with 50 eggs in each group. The A, B and C group eggs were injected with 5μl of pL-2964OVtPAGFP, pL-OVtPA, DMEM and only hole (window) was made in group D, whereas group E was the control group. It was found that the hatchability of group A, B, C, D and E was 10%,18%,24%,46%,94%, dead embryo were 14%,26%, 34%,26%, and 0.02% and eggs without emebryo were 76%,56%,42%,28%, and 0.06% respectively. It was also observed that dead embryo before and after 10 days of incubation were 57.1%,38.5%,29.4% and 15.2% and 42.9%,61.5%,70.6%,84.8% and 1% in group A, B, C, D and E respectively. The dead and live embryo percentage was 24%,44%,58%, 72% and 96% whereas eggs having no embryos 76%,56%,42%,28% and 0.06%. It was found that group D revealed maximum hatchability whereas group A, B and C revealed low hatchablity that may be due to deep injection, aibubbles. It was also observed that dead embryos before 10 days of incubation were more in A, B; C groups whereas after 10 days death ratio was maximum in all groups except control which shows that imporper sealing, heavy contamination casued the embryos death. It is suggested that further research is required and also being continued to observe low hatchablity in injected groups. With further research, the method can be improved and used to produce somatic and germline chimeric chickens efficiently and enable the production of transgenic birds. |
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