S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Agricultural Biotechnology (농생명공학부) Theses (Master's Degree_농생명공학부)
피기백 시스템을 활용한 닭 세포에서의 아비딘, 시스타틴 발현 연구
A study of expression of avidin and cystatin in chicken cells mediated by piggyBac transposition system
- 농업생명과학대학 농생명공학부
- Issue Date
- 서울대학교 대학원
- 학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2015. 8. 한재용.
- Transgenic chicken is an irreplaceable model animal as a bioreactor because of the unique physiological characteristics which differs from mammals. Modern layer hens produce more than 330 eggs per years, which contains 6.5g of proteins. More significant advantage of the use of chicken as bioreactors, in comparison with other mammalian species, is the relatively short generation time of approximately 20 weeks. Thus, the transgenic chicken line can be built up within a comparatively shorter time. Moreover, simple composition of the egg white proteins offers additional merits on the purification process. To take advantage of using chicken as a bioreactor, development of efficient genetic modification technology in chicken system should be preceded for further improvement on avian bioreactor. As an emerging safety issues, non-viral system to produce transgenic chicken has become important.
Biologically active substances contained in egg white have drawn much attention of researchers. Avidin and cystatin are trace components of egg white, in which each makes up 0.05% of the total egg white protein. Both proteins are widely used in the fields of biotechnology and clinical researches
avidin mainly for avidin-biotin technology and cystatin mainly for disease treatment and diagnostics, however, the low contents of avidin and cystatin in natural resources limit their applications. For commercial use, two proteins have been produced from diverse bioreactor systems, including E.coli, Pichia Pastoris and human cell lines. However, the safety issues and cost-effectiveness hinder practical applications of avidin and cystatin. Thus, in this study, we generate founders of transgenic chicken to produce more avidin and cystatin C with the non-viral transposon method.
For efficient gene insertion into the chicken genome, piggyBac transposon vector was modified to overexpress chicken avidin, human cystatin C and chicken cystatin. For higher expression, self-cleaving 2A peptide were inserted between two coding sequences (CDS) of each genes. These expression vectors were expected to produce more protein than expression vectors with one copy of genes. Particularly, codon of human cystatin with 2A peptide gene was optimized for expression in the hen. To confirm the working of constructed vectors, we transfected DF-1 chicken fibroblast cell lines with 6 types of vectors, respectively. After transfection, DF-1 cells were selected with G418 for up to 4 weeks, and RT-PCR for each cell lines were followed. As a result, we verified that the piggyBac transposon system is able to introduce transgenes into chicken genome effectively.
After confirming successful transgene integration into chicken genome using piggyBac system, transgenes were introduced into chicken primordial germ cell (PGC), which is the precursor of functional gamete. Selection with G418 and RT-PCR were performed in the same manner as in DF-1 cell line to demonstrate integration and expression of chicken avidin, human cystatin, and chicken cystatin, respectively. Additionally, RT-PCR analysis was conducted to examine whether G418-selected PGCs preserved the germ-cell properties. The result indicated that G418-selected chicken PGCs expressed transgene without losing germ cell characteristics.
For generating transgenic chicken, transgene-expressing PGCs were transplanted into Korean Oge chicken (KO) recipient embryonic blood vessels. After hatching and sexual maturation, we collected sperm from male founders, and conducted PCR to detect donor PGCs-derived sperm in male founders. From the result of PCR, we detected a total of 17 founders. However, until now, none of founders was confirmed as a germline chimera, which can produce transgenic offspring, via testcross analysis.
In our research, founders for transgenic chicken expressing avidin and cystatin were generated using non-viral piggyBac transposon system. This research can contribute to the studies on avian transgenesis, and provide efficient production system for various biomaterials to overcome the limitations of the conventional system.