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頁籤選單縮合
題名 | 反轉錄病毒起動子捕捉載體之製造 |
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作 者 | 王秀觀; 李桂楨; 張雯; | 書刊名 | 師大生物學報 |
卷期 | 30:1 1995.10[民84.10] |
頁次 | 頁23-29 |
分類號 | 361.8 |
關鍵詞 | 反轉錄病毒; 起動子捕捉; Retrovirus; Promoter trap; |
語文 | 中文(Chinese) |
中文摘要 | 本實驗之目的,在改良反轉錄病毒起動子捕捉載體,以期更有效率的捕捉細胞基因的起動子。U3βGeo/supF質體3端不帶有促進子的LTR的U3區插入了在同一解讀架構上的β-galactosidase (ß -gal)和neomycin phosphotransferase (neo)的雜合標識基因(簡稱ß -geo)。我們將U3 ß –geo/supF質體3端U3區之起動子序列切除,得到ΔU3β-geo/supF質體,以去除起始於5端LTR而終止於3端LTR的轉錄,進而提高起動子捕捉的效率。此外,我們以pBR322質體之複製起源點(ori)及β-lactamase基因(amp)置換ΔU3β-geo/supF質體之supF基因,得到ΔU3β-geo/ori-amp質體,此法有利於病毒插入處鄰近細胞序列的選殖。將各重組質體轉移入ψ2細胞,得到重組病毒製造細胞株,其病毒價數,可籍感染NIH3T3細胞來測定。U3β-geo/supF重組病毒感染之細胞生長良好、緻密,有較多大型細胞株形成。切除U3區之起動子的重組病毒感染之細胞則生長緩慢、不緻密,大型細胞株很少。U3β-geo/supF重組病毒價數與其他已知之鼠類白血病毒價數相近,故推論在LTR上插入3.9kbβ-geo基因並不會影響病毒的複製、插入或包裝。ΔU3β-geo/ori-amp重組病毒價數則降低50%。故推論pBR322質體之ori-amp基因上的有毒序列可能會影響病毒的複製或插入,或者ori/-amp基因之置入,使病毒增長2.1Kb,可能造成病毒包裝的困難而導致病毒價數的降低。 |
英文摘要 | We examined a retrovirus promoter-trap vector that selects for integration in expressed and/or regulated genes. The U3 β-geo/supF vector has a 3.9-kbβ-galactosidase-neomycinphosphotransferase (β-geo) in-frame fusion gene inserted into the 3' LTR of enhancerless Moloney murine leukemia provirus. To eliminateβ-geo transcript that initiates at 5' LTR, U3β-geo/supF vector was modified by deleting 100-bp U3 promoter sequences to generate ΔU3β-geo/supF. In addition, the plasmid pBR322 replication origin andβ-lactamase gene were inserted into gag coding sequence to generateΔU3β-geo/ori-amp that the nearby flanking cellular sequences can be cloned rapidly. Virus producing cells were generated by transfecting plasmid vector into ψ 2 cells. Viruses recovered from cloned producer lines were titered on NIH 3T3 cells, and selected in G418 medium. The cells infected with U3 virus grew well, and a number of large cell clones were observed, whereasΔU3 virus infected cell clones grew slower and a few large cell clones were observed. Titer from U3β-geo/supF was similar to that of other MoMuLV vectors reported. The results indicate that insertion of 3.9-kbβ-geo gene into LTR does not interfere with virus replication, integration or packaging. A 50% decrease of the titer from ΔU3β-geo/ori-amp were observed. The results suggest that 2.3-kb pBR322 replication origin andβ-lactamase gene insertion into gag may interfere with virus replication or integration. Alternatively, a 2.1-kb increase in virus size during replacing supF with ori-amp may also interfere with virus packaging that resulted in titer decrease. |
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