Self-Inactivating MMLV Retrovirus 區聽Gene Expression Vector

The MMLV retroviral ve些雪ctor system is an 山姐efficient vehicle fo化靜r introducing g女信enes permanently into mammalian 做謝cells. It became 市行particularly popular as a gene 女開delivery method for making iP員吧S cells.

MMLV retroviral vectors ar是服e derived from Moloney murine 靜員leukemia virus, which is a membe學行r of the retrovirus family兵為. Wildtype MMLV virus has a plus-strand鄉要 linear RNA genome.

While our wildtype MMLV retrovi兵年rus gene expression內樹 vector utilizes the u工服biquitous promoter functi我舊on in the 5' long ter購話minal repeat (LTR信匠) of wildtype MMLV genome for driving 他人expression of the愛答 gene of interest (GOI), the self坐嗎-inactivating MMLV retroviru現短s gene expression vector不拿 allows users to 北看select any promoter of their c費子hoice for driving 著子GOI expression. This is achieved by下讀 the deletion of錯森 the U3 region in the MMLV 3’ LTR 低月which self-inactivates鐵讀 the promoter activity in the 5' LTR by麗兵 a copying mechanism during 錢能viral genome integration. This not還亮 only provides users with the flexibil中鐵ity to add their p學服romoter of choice年師 for driving GOI expression bu個問t also eliminates the ri文工sk of oncogenic 家遠activation of adja公書cent genes upon vector integratio雜間n, thereby enabling such vector銀日s to have a higher safety profi在身le compared to wildtype MMLV術體 vectors.

A self-inactivating MMLV retroviral 些輛vector is first constructed as a plasm喝來id in E. coli. It is then trans人花fected into packaging cells alo明樂ng with several helper plasmi算些ds. Inside the packaging ce海身lls, vector DNA located between白下 the LTRs is transcr場可ibed into RNA, and viral pr知山oteins expressed by the h上吧elper plasmids fu票廠rther package the RNA into virus. Live少樂 virus is then released int好書o the supernatant, which can雨吃 be used to inf熱學ect target cells林員 directly or after concent樂劇ration.

When the virus is added to targe司務t cells, the RNA cargo is shutt務唱led into cells where it is會相 reverse transcribed 子黑into DNA and randomly integra長舊ted in the host genome. Any ge船藍ne(s) that were placed in-between the t坐中wo LTRs during ve得聽ctor cloning are pe慢票rmanently inserted into h拿討ost DNA alongside the rest of viral gen高市ome.

By design, self-inactivating MMLV靜黃 retroviral vectors lack the gen視麗es required for viral 金樹packaging and transduction (these ge答他nes are carried by helper plasmids 有錢or integrated into packaging cells in雪電stead). As a result, viruses p看業roduced from these vectors ha錢廠ve the important safety fea黃木ture of being r小但eplication incompetent西什 (meaning that they can transdu她暗ce target cells but cannot replicate i下我n them).

For further information about th上個is vector syste什動m, please refer to the papers be術吧low.

Permanent integration of vector但西 DNA: Conventional transfection resul身靜ts in almost entirely 為火transient delivery of DNA into 服窗host cells due 懂呢to the loss of DNA over time. This 錢錯problem is especially prominent in ra和農pidly dividing cells. In contr男鐘ast, retroviral transduction 話照can deliver genes permanently into 的火host cells due to integration of the vi子呢ral vector into the host genome.

Broad tropism: Our packaging system友計 adds the VSV-G e知看nvelop protein to the 都但viral surface. This protein has br微農oad tropism. As a 術相result, cells from all commonly used ma但聽mmalian species such as human, mous笑聽e and rat can be transduced. Furt高員hermore, many cell types can be離腦 transduced, though our 愛計vector has difficulty transducing 拍在non-dividing cells (see d外話isadvantages below).

Customizable internal promoter: Our vector is designed to self-inac銀信tivate the promoter activity in it了舞s 5' LTR upon integrat我吃ion into the genome. As a 家畫result, users ca河家n put in their own promoter to dr煙你ive their GOI within t老去he vector. This is a disti一刀nct advantage over our wildtype MMLV下作 retrovirus vectors, which rely on 身花the promoter fun拿舞ction of 5' LTR t朋技o drive the ubi民煙quitous expression of the GO工錢I.

Relative unifor話鐵mity of gene delivery: Generally, viral購睡 transduction can del分山iver vectors into 如時cells in a relatively uniform manner. I通從n contrast, conventional物那 transfection of plasmid vectors can下日 be highly non-unif你報orm, with some cells r愛河eceiving a lot of copies while 算還other cells receiving few copies or爸多 none.

Effectiveness in vi亮藍tro and in vivo: While our vector這年 is mostly used for i有遠n vitro transduction of cultured月火 cells, it can also be used to tr樂哥ansduce cells in live a鐵麗nimals.

Safety: The safety of our vector is 放近ensured by two features. One is th購樂e partition of genes required 拿舊for viral packaging and熱慢 transduction into several helper pl雜市asmids; the other is self-inactiva外拍tion of the promoter activity in th國要e 5' LTR upon ve身討ctor integration. As她做 a result, it is們錢 essentially impossible for 區日replication competent virus 見喝to emerge during packaging and transdu小土ction. The health risk of 懂光working with our vector is海玩 therefore minimal.