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題名 | 利用第二型腺病毒伴隨病毒載體轉染建立攜有綠螢光蛋白質與新黴素耐受基因之小鼠胎體成纖維細胞株=Transfection of Mouse Embryonic Fibroblast Cell Line with GFP and Neomycin Resistant Genes by Adeno-associated Viral Vector Type 2 |
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作者姓名(中文) | 楊鎮榮; 廖家信; 薛佑玲; 陳立人; | 書刊名 | 畜產研究 |
卷期 | 39:4 民95.12 |
頁次 | 頁289-299 |
分類號 | 437.21 |
關鍵詞 | 第二型腺病毒伴隨病毒載體; 綠色螢光蛋白質; 新黴素耐受基因; STO小鼠胎體成纖維細胞; Adeno-associated viral vector type 2; Green fluorescent protein; Neomycin resistant gene; Mouse embryonic fibroblasts; |
語文 | 中文(Chinese) |
中文摘要 | 本研究之目的係利用第二型線病毒伴隨病慣載體(adeno-associated viral vector type 2, AAV2)進行STO小鼠胎體成纖維細胞(STO mouse embryonic fibroblasts)進行基因轉殖,期建立攜有外源綠螢光(green fluorescent protein, GFP))基因及新黴素耐受基因(neomycin resistant resistant gene, Neo')之STO小鼠胎體成纖維細胞株,以做為豬胚幹細胞之供養層細胞。試驗結果顯示,利用G418進行STO細胞之篩選試驗,其最適篩選濃度為400g/ml,其篩選7天後的死亡率為41.8±11.6%。利用AAV2載體轉染STO小鼠胎體成纖維細胞後,轉染率可達50.9±17.1%,利用於培養液中添加400 g/ml的G418進行轉染後篩選,經過4天之繼代篩選後,純度達70.2±12.0%,再配合以流式細胞分析法(flow cytomcetry)進行GFP表現陽性(GFP+)細胞分離,分離後之STO/GFP+小鼠胎體成纖維細胞之純度可達93.6±2.8%。且經過轉染後的STO/GFP+細胞株,用於培養豬胚幹細胞,能維持幹細胞群落之未分化狀態,顯示本STO/GFP+細胞株可做為豬胚幹細胞之供養層細胞。並且,因為本STO/GFP+細胞株攜有外源性GFP報導基因及Neo'基因,未來可作為豬與其他物種的胚幹細胞進行基因轉殖試驗後,以neomycin篩選時之供養層細胞,為胚幹細胞之基因轉殖試驗提供有價值的應用基礎。 |
英文摘要 | The Purpose of this study was to establish a mouse embryonic fibroblast cell line carrying green fluorescent protein (GEP) and neomycin resistant (Neo') genes. Murine embryonic fibroblasts from STO cell line were transfected with adeno-associated viral vector type 2 (AAV2) followed by being cultured in G418 selection medicum. The results showed that the optimal concentration of G418 selection was 400μg/ml and its mortality was 41.8±11.6% after 7 d of selection. The transfection rate of STO cells transfected with AAV2 was 50.9±17.1%, as determined by GFP expression. Following the selection with G418-containing medium (400μg/ml) and flowcytometry sorting, the proportion of GFP-expressing STO cells was promoted to 70.2±12.0% and 96.6±2.8%, respectively. The transfected STO/GFP+ cell line could support the maintenance of undifferentiated porcine embryonic stem cells. This STO/GFP+ cell line which carried Neo' gene might provide for a valuable source of feeder layer for the cultivation and selection of transgenic ES cells, which were transfected with Neo' gene as a selection marker. |
本系統之摘要資訊系依該期刊論文摘要之資訊為主。