哺乳動物基因FLEX條件性表達慢這坐病毒載體（Cre-On）

FLEX條件性表達慢病毒載體結合了載體家的高效的第三樹事代慢病毒載體系統和Cre響應的FLE匠我X條件性基因表達載體，幫助您實現在病毒宿主基因組上永久整合Cre重組酶響應的市有FLEX Cre-On基因調控開(kāi)關。有公FLEX Cre-On系統利用位于目的基因兩鐵嗎(liǎng)側的Lox-變體重組位點，在Cre重可銀組酶的調控下目的基因發(fā)生反樹什轉，目的基因正常表達；而Cre不存在的情況下，目的基因不表舞匠達。

FLEX Cre-On系統由兩(liǎn你自g)對(duì)異型Lox-變體重組位點組成(chéng)，其中野生型序列稱看到爲LoxP，突變體稱爲Lox2272，分别位見車于反向(xiàng)基因ORF（相月理對(duì)啓動子方向(xiàng)而言）的兩(liǎn紙時g)側。 兩(liǎng)種(zhǒng)Lox-變體都(dōu)可被(bè坐媽i)Cre識别，但隻有相同的Lox位點錯讀對(duì)可以彼此重組，與其他Lox-變體不能(néng慢小)進(jìn)行重組。 LoxP和Lox2272位點以交替方式位于ORF兩票吃(liǎng)端，每對(duì)位點互爲反向(xiàng)。Cre重組酶不多著存在的情況時(shí)，目的基因由于其編碼方向(xiàng)與啓動子方向(員火xiàng)相反，所以不發(fā)生表達；在Cre的存在下，朋工LoxP和Lox2272位點對(duì)分别重組，導緻ORF方向(xiàng頻南)反轉爲正向(xiàng)，且其中的一對(duì)重服有組位點將(jiāng)具有相同的方向(xiàng新拍)，Cre會(huì)介導産生正向(xiàng)重組切割，繼而分别留下一站在個不同的重組位點。 ORF的方向(xiàng)反轉，使得目的基因可如生以在啓動子的驅動下正常表達。由這喝于ORF側翼是兩(liǎng)個不同的Lox-變體位點，所以即使就吃存在Cre也不會(huì)進(jìn)一步發(fā)生重組。

慢病毒載體首先以質粒的方式構建信不并使用E. coli擴增，然後(hòu)與多個輔對道助質粒一同轉染至包裝細胞。載體上的兩(l爸低iǎng)個LTR區域之間的DNA序列將(jiāng)被(bèi)轉錄成(c藍了héng)RNA，而輔助質粒表理這達的病毒蛋白進(jìn)一步與這(zhè)些RNA組裝形成(ché影河ng)完整的病毒顆粒。有活性的病毒顆粒被事好(bèi)釋放到上清液中。病毒上生東清液可直接轉染或者濃縮後(hòu)轉染請技細胞。

當病毒感染靶細胞時(shí)，唱從病毒RNA被(bèi)反轉錄成(chéng)DNA，然後(hòu)永久都用性整合到宿主基因組。位于兩(liǎng)個LTR區他拿域之間的FLEX Cre-On開(kāi)關連同其外呢他病毒基因組組分永久整合到宿主基因組。目的基因的激活表達可以在Cre重組酶的介年北導下通過(guò)反轉ORF編碼方向呢要(xiàng)實現。

通過(guò)改造優化，我們的慢病毒載體删除了與病毒包裝和轉導相關的基兵數因（這(zhè)些基因由輔助質粒進(jìn)行表達，用于病毒車請包裝過(guò)程），使産生的慢病毒顆粒是複制缺陷型的。即長不包裝的病毒隻具有轉導靶細胞的能(néng)力，而無法在靶細胞中進(jì又習n)行大量複制，因而具有很高的生物安全性。

關于該載體系統的更多信息，請參考以下文獻司廠。

哺乳動物基因FLEX條件性表達慢病毒載體有村（Cre-On）專爲在哺乳動物細胞和動物體内實現高效火小的條件性基因激活表達而設計。目的基因剛開(kāi)始時(shí)爲默地離認不表達，但是在Cre重組酶共表達的情況下將(jiāng)被(bè校身i)激活表達，并且將(jiāng)反轉和改變目山我的基因的編碼方向(xiàng)。Cre存在的情況下，目的基因中森的表達受用戶自定義的啓動子的調控。

我們目前采用的是第三代慢病毒包裝載體系統。經(jīng)優化，該載體在大火我腸杆菌體内具有很高的拷貝數，包裝的活病毒具有很高的滴度，對(duì)大美快多數宿主細胞具有高效的轉導能(néng)力，能(né湖工ng)有效地把載體整合到靶細胞有機基因組并實現外源基因的高水平表達。

RSV promoter:&n信河bsp;Rous sarcoma virus promoter. 光謝It drives trans空懂cription of viral RNA in p說們ackaging cells. This RNA is then pack章照aged into live virus.

5' LTR-ΔU3: A deleted version of the HIV-1 5' lo船老ng terminal repeat.事術 In wildtype lentivir器妹us, 5' LTR and 3' LTR are 冷器essentially identical in sequence風我. They reside on two end電術s of the viral genome and point in th朋用e same direction. Upon vira票算l integration, the 3可車' LTR sequence is copied onto t年錢he 5' LTR. The LTRs carry bot商笑h promoter and polyadenyl離算ation function, such that in wildtype v放放irus, the 5' LTR acts as一要 a promoter to drive花區 the transcription 友家of the viral gen也中ome, while the 3' LTR acts as a p煙不olyadenylation signa市男l to terminate the ups吧和tream transcript. On our劇筆 vector, 5' LTR-Δ但些U3 is deleted for a r睡習egion that is required for the LTR's p人知romoter activity normally f民算acilitated by the viral transcription f微紅actor Tat. This does not affect the p資明roduction of viral RN麗說A during packaging becau短務se the promoter func下下tion is supplemented by the RSV pro技多moter engineered做民 upstream of 5'LTR-也飛ΔU3 LTR.

Ψ: HIV-1 packaging signal requi拍業red for the packaging of viral RN吧鄉A into virus.

RRE: HIV-1 Rev respo員光nse element. It allows t不務he nuclear export 照姐of viral RNA by the viral Rev pr長廠otein during vi又坐ral packaging.

cPPT: HIV-1 Central 頻民polypurine tract. It creates a "DNA個銀 flap" that increases nuclear import 匠員of the viral genome during target費請 cell infection. This improve人劇s vector integration into the離報 host genome, resu這體lting in higher transduction efficie讀弟ncy.

Promoter: The promoter driving海通 your gene of interest is placed here.計朋

Lox2272: Recombina關關tion site for Cre 動農recombinase. Mutated Lox site with two 懂很base substitutions of wild type LoxP秒說. Incompatible with LoxP sites章鐵. When Cre is present, th師器e LoxP and LoxP227低這2 sites will be cut and r是藍ecombine with compatible sites事為.

LoxP: Recombination site for C行制re recombinase. Incompat森冷ible with Lox2272 sites. When C東朋re is present, the LoxP and Lox22們訊72 sites will be cut理做 and recombine with com路分patible sites.

ORF: The open reading錯南 frame of your gene of inter裡筆est is placed here, in an ant去行isense orientation.

WPRE: Woodchuck hep跳黃atitis virus posttransc會呢riptional regulatory element. It enhan舞南ces transcriptional termination離村 in the 3' LTR during viral RNA 快花transcription, which leads to higher電書 levels of functional遠綠 viral RNA in packaging cells and h妹短ence greater viral titer. 計呢It also enhances transcriptional t請自ermination during th計唱e transcription of t睡電he user's gene of interest 下他on the vector, leading to their high藍理er expression levels.

CMV promoter: Human cytomegalovirus immed暗公iate early promoter. It drives the ubi懂影quitous expression of the downst在大ream marker gene.

Marker: A drug selection gene (s光東uch as neomycin resistance), a visual作愛ly detectable gene 費內(such as EGFP), or a dual道筆-reporter gene (such as EG聽都FP/Neo). This allows ce近制lls transduced with the v男報ector to be selected and/or visuali自雨zed.

3' LTR-ΔU3: A truncated version of吃算 the HIV-1 3' long ter要工minal repeat that deletes th聽吃e U3 region. This靜術 leads to the self-ina動器ctivation of the promoter activit男員y of the 5' LTR upon viral vector in資討tegration into the host genome 作國(since the 3' LT制男R is copied onto 5' LTR during viral i筆路ntegration). The polyadenylation 國人signal contained 內習in 3' LTR-ΔU3 serves to ter雪外minates all upstr頻笑eam transcripts produce喝化d both during viral pa和麗ckaging and afte下是r viral integration into the host geno我朋me.

SV40 early pA: Simian virus 40 early polyadenylation 路問signal. It furthe鐵可r facilitates transcri少視ptional termination after the 3' LTR船笑 during viral RNA transc線文ription during packagin房和g. This elevates the level of友說 functional viral RNA知海 in packaging cells, t窗電hus improving viral titer.

Ampicillin: Ampicillin resistance gene. It allo秒不ws the plasmid to be mainta科體ined by ampicillin selection in E. col錯訊i.

pUC ori: pUC origin of replication. Pla筆山smids carrying this origin exist in什路 high copy numbers 讀不in E. coli.

載體容量受限：野生型的慢病毒基因組大小約爲9.2 kb，而在我們的慢病毒載體中，病毒包煙西裝和轉導的必要元件約爲2.8 kb，餘下文笑6.4 kb的空間容納客戶的目的序列。當病毒載體超過(guò)以上大小限制，數懂病毒的包裝滴度將(jiāng)會(huì)間從大大降低。我們的慢病毒載體除了可以插入靶基因的序列外，還(h會票ái)可以插入啓動子和篩選标記等載體元件。如果目的坐器基因和這(zhè)些載體元件長(cháng)度超過(g月朋uò)了6.4 kb，病毒的産量有可能(néng)會(huì)明顯下風來降。

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(hò冷師u)測定病毒滴度。因此慢病毒轉染相對(duì)于常規質粒轉染，技術難度兵術更高，周期更長(cháng)。