哺乳動物基因表達慢病毒載體

我們目前采用的是第三代慢病毒包裝載體系統。經(jīn光紅g)優化，該載體在大腸杆菌體内具有很高的拷貝數，包裝的活病車文毒具有很高的滴度，對(duì)大懂近多數宿主細胞具有高效的轉導能(néng)少街力，能(néng)有效地把載體整合到靶細胞基因組并實現外源基因的裡空高水平表達。

載體容量受限：野生型的慢病毒基因組大小約爲9.2 kb，而在我們的慢病毒時兒載體中，病毒包裝和轉導的必要元件約爲2.8 kb，餘下6.4 kb的空間容納路南客戶的目的序列。當病毒載體超過(guò)以上大小限制，病毒的包裝滴度將(ji厭在āng)會(huì)大大降低。我們的慢病毒載體除個學了可以插入靶基因的序列外，還(hái)可以插車光入啓動子和篩選标記等載體元件。如果目的基因頻影和這(zhè)些載體元件長(cháng)度超過(guò)了6.4 黃煙kb，病毒的産量有可能(néng)會(huì從購)明顯下降。

技術複雜：使用慢病毒載體時(shí)，需要在包裝細胞中産生活病毒，然後(hòu)測定病費下毒滴度。因此慢病毒轉染相對(duì)于常規質粒轉染，技術難度更高，周視器期更長(cháng)。

RSV promoter: Rous sarcoma virus promoter. It d冷拍rives transcription o綠到f viral RNA in packaging cells. Th都友is RNA is then packaged into live virus冷光.

Δ5' LTR: A deleted version of the雨問 HIV-1 5' long terminal repea鄉飛t. In wildtype lentiv票這irus, 5' LTR and 3' LTR are essentially到媽 identical in sequ吃跳ence. They resi視照de on two ends of the viral gen作資ome and point in the same direct吧都ion. Upon viral int分商egration, the 3' LTR seque費服nce is copied onto the 5得那' LTR. The LTRs car多計ry both promoter 讀到and polyadenylation function, such t件體hat in wildtype virus, the 5年生' LTR acts as a還多 promoter to drive the trans雨看cription of the viral genom銀頻e, while the 3' LTR acts as a 火友polyadenylation signal to如如 terminate the upstream t風劇ranscript. On our vec開坐tor, Δ5' LTR is 兵有deleted for a region that is r錯明equired for the LTR風是's promoter activity normally fac行門ilitated by the viral tran些知scription factor Tat. This d答花oes not affect the 讀關production of vi亮一ral RNA during packaging beca火票use the promoter function i熱東s supplemented by the RSV promo玩短ter engineered upstream 廠歌of Δ5' LTR.

Ψ: HIV-1 packaging sig用視nal required for the p遠地ackaging of viral RNA into v樂船irus.

RRE: HIV-1 Rev response element. It allow到錯s the nuclear ex工舞port of viral RNA by the viral Rev prot日兵ein during vira下長l packaging.

cPPT: HIV-1 Central polypurine tr照但act. It creates a "DNA flap" tha友物t increases nuclear i兒不mportation of the viral geno長那me during target cell infe兵家ction. This improves vector in吧光tegration into the h業路ost genome, resulting in higher transdu海笑ction efficiency音兵.

Promoter: The promoter driving your gene of int不知erest is placed here.

Kozak: Kozak consensus sequence. It i房看s placed in front of the start codon 放報of the ORF of int制船erest because it is beli自鐵eved to facilitate translation ini子很tiation in eukaryotes.

ORF: The open reading frame of 對慢your gene of interest is placed her門他e.

WPRE: Woodchuck hepatitis virus posttranscrip玩的tional regulator紙理y element. It e飛門nhances viral RNA stabil懂愛ity in packaging 唱我cells, leading t民中o higher titer of packag信購ed virus.

mPGK promoter: Mouse phosphoglycerate kinase 1去弟 gene promoter. It drives t線業he ubiquitous exp在秒ression the downstream marker gene.

Marker: A drug selection gene (such as neom化現ycin resistance), a visually detectable得費 gene (such as EGFP), or a dual-reporte能玩r gene (such as EGFP/Neo). This低是 allows cells transduced with the vecto民了r to be selected and/or vis水南ualized.

ΔU3/3' LTR: A truncated version of the HIV-1 3' 金說long terminal repeat近行 that deletes th風家e U3 region. This leads t鐵書o the self-inacti服友vation of the promoter activ多嗎ity of the 5' LTR 亮信upon viral vector inte章跳gration into the host genome (due 章大to the fact that 3' LTR is copied onto 開姐5' LTR during viral integration). 老離The polyadenylation sign請些al contained in ΔU3/3' L爸水TR serves to terminates all upst好校ream transcripts produced both dur短師ing viral packaging and afte為雪r viral integration into知街 the host genome.

SV40 early pA: Simian virus 40 early po事路lyadenylation signal. It furt鐘朋her facilitates transcription微照al termination afte子件r the 3' LTR during viral RNA tr光化anscription during packa從業ging. This elevates the level of fun鄉頻ctional viral RNA in packaging cell白明s, thus improving vi從我ral titer.

Ampicillin: Ampicillin resistance gene. It離愛 allows the plasmid to be maintained by文相 ampicillin sel低空ection in E. coli.

pUC ori: pUC origin of replication. Plasmi朋計ds carrying thi購行s origin exist in high copy num關綠bers in E. coli.