反轉錄病毒起動子捕捉載體之製造

Abstract

本實驗的目的，在改良反轉錄病毒起動子捕捉載體，以其更有效率的捕捉細胞基因的起動子。U3β-geo/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-galactosidase(β -gal)和neomycmphosphotransferase (neo)的雜合標識基因(簡稱3 -geo)。我們將u3 β -geo/supF質體3端U3區之起動子序列切除，得到L1 u3 β -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質體支複製起原點(ort)及β-lactamase基因(amp)置換 U3β -geo/supF質體之supF基因，，得到L1 U3β -ge%ri-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2 細胞，得到重組病毒製造細胞株，其病毒價數，可將感染NIH3T3細胞來測定。U3β -geo/supF 重負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。U3 β-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插入3.9-kb β-geo基因並不會影響病毒的複製 插入或包裝 oU3 β –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2.1 Kb，可能造成病毒包裝的的難而導致病毒價數的降低。

We examined a retrovirus promoter-trap vector that selects for integration in expressed and/or reb'lllated genes. The U3/3 -gcu!supF ,,,,etor has a 3,9-kb /3-galactosidase-neomycinphosphotransferase (/3 -gco) in-frame1,.. ,011 gre mserted into the 3' LTR of enhancerless Moloney murine leukemia provirus, To eliminate /3 –gco transcript that initiates at 5' LTR, U3/3 -geo/supF vector was modill~d by deleting 100-bp U3 promoter sequences generate L1 U3 /3 -geo/supF, In addition, the plasmid pBR322 replication origin and /3 -lactamase gene were ia~eited mto gag coding sequence to generate L1 U3/3 -ge%ri-amp that the nearby flankilg cellular sequences can be cloned rapidly, Virus producing cells were generated by transfecting plasmid vector into t/J 2 cells, Viruses recovered from cloned producer lines were titered on NIH 31'3 cells, and selected in G418 medium, The cells infected with U3 virus grew well, and a number of large cell clones were observed, whereas L1 U3 virus infected cell clones grew slower and a few large cell clones were observed, Titer from U3 /3 -geo/supF was similar to that of other MoMuLV vectors reported, The results indicate that insertion of 3,9-kb /3 -geo gene into LTR does not mterfere with virus replication, integration or packaging, A 50% decrease of the titer from L1 U3 /3 -ge%ri-amp were observed, The results suggest that 2,3-kb pBR322 replication origin and /3 -lactamase gene insertion into gag may interfere with virus replication or inteb'fation, Alternatively, a 2,I-kb increase in virus size during replacing slipF with ori-amp may also interfere with virus packaging that resulted in titer decrease.