幾丁聚醣/DNA奈米微粒於基因轉殖雞之應用

Abstract

本研究發展出新的方法製得分散性良好且粒徑於100-200 nm之間的 幾丁聚醣/DNA 奈米微粒。經由幾丁聚醣與三磷酸鈉之間的離子膠聯反應，彼此間於適當的濃度配比下，可生成分散性良好且粒徑在100 nm以下的幾丁聚醣奈米微粒，同時，我們進一步利用成核反應與界面電位說明奈米微粒生成的機制。在質量比為10/1的條件下，幾丁聚醣可有效包覆DNA形成 幾丁聚醣/DNA 奈米微粒，佐以雷射奈米粒徑暨界面電位量測儀、穿透式電子顯微鏡、掃瞄式電子顯微鏡證實此奈米微粒的粒徑與穩定性，且經由DNAse І的消化作用說明幾丁聚醣具有保護DNA的能力。於293HEK細胞的轉染實驗中，幾丁聚醣/DNA 奈米微粒具有47.53 %的轉染效率;雖然lipofectamineTM 2000具有87.7 %的轉染效率，但是細胞活性則遠低於 幾丁聚醣/DNA 奈米微粒。於轉染雞胚胎的實驗中，經由幾丁聚醣轉染的蛋之孵化率遠高於lipofectamineTM 2000。雖然於gDNA的PCR產物中沒有檢測到預期的特異片段，不過良好生物相容性的優勢若搭配其他轉殖策略，相信於未來對基因轉殖雞之研究極具發展潛力。

In this study, preparation of well-dispersed chitosan/DNA nanoparticles with size of 100-200 nm was developed. By ionic gelation between chitosan and sodium tripolyphosphate, chitosan nanoparticles with size below 100 nm were prepared under adequate concentration. Furthermore, the mechanism of chitosan nanoparticles formation was expounded by nucleation theory and zetapotential phenomenon. Chitosan/DNA by 10/1 in mass ratio, DNA could be encapsulated effectively in the nanoparticles; meanwhile, size analysis was performed under dynamic light scattering, TEM, and SEM. The stability of the nanoparticles was proven by zetapential analysis. DNA encapsulated in the nanoparticles could be protected from DNAseⅠ degradation. In 293 HEK cells transfection experiment, efficiencies of chitosan/DNA nanoparticles and lipofectamineTM 2000-DNA were 47.53 % and 87.8 % respectively. The cell viability of lipofectamineTM 2000-DNA was much lower than that of chitosan/DNA nanoparticles. The egg hatchability after chitosan/DNA nanoparticles transfection was far better than that of lipofectamineTM 2000-DNA transfection one. Although genomic DNA was not detected in all chicks, if the advantage of good biocompatibility of chitosan/DNA nanoparticles is cooperated with other powerful transgenic techniques or strategies, chitosan/DNA nanoparticles will have great potential in generating transgenic chicken.