LEX條件性表達(dá)慢病毒載體結(jié)合了載體家的高效的第三代慢病毒載體系統(tǒng)和Cre響應(yīng)的FLEX條件性基因表達(dá)載體，可以幫助您實(shí)現(xiàn)在多種多樣的哺乳動(dòng)物細(xì)胞中以慢病毒轉(zhuǎn)導(dǎo)Cre響應(yīng)的FLEX基因表達(dá)調(diào)控開(kāi)關(guān)。FLEX Cre-Switch系統(tǒng)的兩個(gè)Lox-變體重組位點(diǎn)之間包含一對(duì)反向平行的ORF，在Cre重組酶的調(diào)控下ORF上的兩個(gè)目的基因編碼方向反轉(zhuǎn)，從而讓一個(gè)目的基因從激活表達(dá)變成沉默，另一個(gè)目的基因從沉默變成激活表達(dá)。

FLEX Cre-switch系統(tǒng)由兩對(duì)異型Lox-變體重組位點(diǎn)組成，其中野生型序列稱為L(zhǎng)oxP，突變體稱為L(zhǎng)ox2272，分別位于兩個(gè)基因ORF的兩側(cè)。 兩個(gè)ORF相互反向，其中一個(gè)ORF具有正確的讀碼方向（相對(duì)于啟動(dòng)子）。兩種Lox-變體都可被Cre識(shí)別，但只有相同的Lox位點(diǎn)對(duì)可以彼此重組，與其他Lox-變體不能進(jìn)行重組。LoxP和Lox2272位點(diǎn)以交替方式位于兩個(gè)ORF兩端，每對(duì)位點(diǎn)互為反向。Cre重組酶不存在的情況時(shí)，第一個(gè)ORF由于其編碼方向與啟動(dòng)子方向相同，可以在客戶定制的啟動(dòng)子驅(qū)動(dòng)下正常表達(dá)，而第二個(gè)ORF由于其方向相反則無(wú)法正常表達(dá)。在Cre的存在下，LoxP和Lox2272位點(diǎn)對(duì)分別重組，導(dǎo)致兩個(gè)ORF方向反轉(zhuǎn)，且其中的一對(duì)重組位點(diǎn)將具有相同的方向，Cre會(huì)介導(dǎo)產(chǎn)生正向重組切割，繼而分別留下一個(gè)不同的重組位點(diǎn)。 ORF方向的反轉(zhuǎn)，使得第二個(gè)OR可以在啟動(dòng)子的驅(qū)動(dòng)下正常表達(dá)，而第一個(gè)ORF將不表達(dá)。 由于ORF側(cè)翼是兩個(gè)不同的Lox-變體位點(diǎn)，所以即使存在Cre也不會(huì)進(jìn)一步發(fā)生重組。

慢病毒載體首先以質(zhì)粒的方式構(gòu)建并使用E. coli擴(kuò)增，然后與多個(gè)輔助質(zhì)粒一同轉(zhuǎn)染至包裝細(xì)胞。載體上的兩個(gè)LTR區(qū)域之間的DNA序列將被轉(zhuǎn)錄成RNA，而輔助質(zhì)粒表達(dá)的病毒蛋白進(jìn)一步與這些RNA組裝形成完整的病毒顆粒。有活性的病毒顆粒被釋放到上清液中。病毒上清液可直接轉(zhuǎn)染或者濃縮后轉(zhuǎn)染細(xì)胞。

當(dāng)慢病毒感染靶細(xì)胞時(shí)，病毒RNA被反轉(zhuǎn)錄成DNA，然后永久性整合到宿主基因組。位于兩個(gè)LTR區(qū)域之間的載體組分永久整合到宿主基因組。對(duì)于上述的FLEX介導(dǎo)的Cre-Switch條件性表達(dá)慢病毒載體，F(xiàn)LEX Cre-Switch調(diào)控的兩個(gè)反向平行的ORF位于兩個(gè)LTR之間的區(qū)域。這兩個(gè)ORF也被永久整合到宿主基因組當(dāng)中，并在Cre調(diào)控下使得其中一個(gè)ORF獲得激活表達(dá)，另一個(gè)則表達(dá)失活。

關(guān)于該載體系統(tǒng)的更多信息，請(qǐng)參考以下文獻(xiàn)。

FLEX Cre-Switch條件性表達(dá)慢病毒載體專為在哺乳動(dòng)物細(xì)胞和動(dòng)物體內(nèi)實(shí)現(xiàn)兩個(gè)ORF可切換的基因表達(dá)而設(shè)計(jì)。目的基因表達(dá)受到用戶自定義的啟動(dòng)子調(diào)節(jié)，并在Cre重組酶共表達(dá)的情況下沉默一個(gè)目的基因，并激活另一個(gè)目的基因表達(dá)。

我們目前采用的是第三代慢病毒包裝載體系統(tǒng)。經(jīng)優(yōu)化，該載體在大腸桿菌體內(nèi)具有很高的拷貝數(shù)，包裝的活病毒具有很高的滴度，對(duì)大多數(shù)宿主細(xì)胞具有高效的轉(zhuǎn)導(dǎo)能力，能有效地把載體整合到靶細(xì)胞基因組并實(shí)現(xiàn)外源基因的高水平表達(dá)。

基因激活失活可控：兩個(gè)ORF的方向彼此相反確保當(dāng)表達(dá)正向方向的ORF時(shí)，反向的ORF被抑制，可防止基因發(fā)生泄漏表達(dá)。

外源基因的穩(wěn)定整合：常規(guī)質(zhì)粒轉(zhuǎn)染只能實(shí)現(xiàn)外源基因的瞬時(shí)表達(dá)，這種外源基因會(huì)隨著宿主細(xì)胞的分裂而不斷丟失，在快速分裂的細(xì)胞中顯得尤為顯著。相反的是，慢病毒轉(zhuǎn)導(dǎo)的目的基因能穩(wěn)定地整合到宿主細(xì)胞的染色體中 ，因而會(huì)隨著宿主細(xì)胞的分裂而穩(wěn)定遺傳。

滴度高：我們的病毒載體可以包裝出高滴度的病毒。我們提供的病毒包裝服務(wù)，病毒滴度可以達(dá)到>10⁹ TU/ml。在這樣的病毒滴度下，如果選擇合適的劑量去轉(zhuǎn)導(dǎo)體外培養(yǎng)的哺乳動(dòng)物細(xì)胞，則轉(zhuǎn)導(dǎo)效率可接近100%。

宿主范圍廣泛：我們的病毒包裝系統(tǒng)包裝出來(lái)的病毒含有VSV-G包膜蛋白，此蛋白擁有非常廣泛的親和性，可以轉(zhuǎn)導(dǎo)幾乎所有的哺乳動(dòng)物細(xì)胞，包括分裂細(xì)胞，非分裂細(xì)胞，原代細(xì)胞，穩(wěn)定細(xì)胞系，干細(xì)胞，分化細(xì)胞，貼壁細(xì)胞和懸浮細(xì)胞等各類哺乳動(dòng)物細(xì)胞，甚至還可以轉(zhuǎn)導(dǎo)一些非哺乳動(dòng)物細(xì)胞。使用傳統(tǒng)的轉(zhuǎn)染方式轉(zhuǎn)導(dǎo)神經(jīng)元細(xì)胞是非常難的，但是采用我們慢病毒載體系統(tǒng)可以輕易的實(shí)現(xiàn)神經(jīng)元細(xì)胞的轉(zhuǎn)導(dǎo)。相對(duì)于在某些細(xì)胞中具有較低轉(zhuǎn)導(dǎo)效率的腺病毒和不能用于非分裂細(xì)胞的逆轉(zhuǎn)錄病毒而言，利用我們的慢病毒包裝系統(tǒng)包裝出來(lái)的病毒具有廣泛的親和性。

基因拷貝數(shù)相對(duì)均一：通常情況下，采用病毒轉(zhuǎn)導(dǎo)的方式可以比較均一的將外源基因轉(zhuǎn)入靶細(xì)胞中，而傳統(tǒng)的質(zhì)粒轉(zhuǎn)染則呈現(xiàn)出較高的不均一性，導(dǎo)致某些細(xì)胞會(huì)獲得較多拷貝質(zhì)粒而某些則會(huì)獲得較少甚至完全沒(méi)有。

體內(nèi)外實(shí)驗(yàn)均有效：我們的載體不僅擁有良好的體外細(xì)胞轉(zhuǎn)導(dǎo)能力，同樣適用于體內(nèi)活體動(dòng)物實(shí)驗(yàn)。

安全性高：我們的病毒載體系統(tǒng)具備了以下兩大特點(diǎn)，因而具有非常高的安全性。一、病毒包裝和轉(zhuǎn)導(dǎo)所必需的基因由三個(gè)輔助質(zhì)粒分開(kāi)表達(dá)。二、5' LTR的啟動(dòng)子自失活。因此，在進(jìn)行病毒包裝和病毒轉(zhuǎn)導(dǎo)的時(shí)候不會(huì)產(chǎn)生具有復(fù)制能力的病毒顆粒，使用我們的載體對(duì)人體的健康威脅也是最低的。

載體容量受限：野生型的慢病毒基因組大小約為9.2 kb，而在我們的慢病毒載體中，病毒包裝和轉(zhuǎn)導(dǎo)的必要元件約為2.8 kb，余下6.4 kb的空間容納客戶的目的序列。當(dāng)病毒載體超過(guò)以上大小限制，病毒的包裝滴度將會(huì)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，還可以插入啟動(dòng)子和篩選標(biāo)記等載體元件。如果目的基因和這些載體元件長(zhǎng)度超過(guò)了6.4 kb，病毒的產(chǎn)量有可能會(huì)明顯下降。

技術(shù)復(fù)雜：使用慢病毒載體時(shí)，需要在包裝細(xì)胞中產(chǎn)生活病毒，然后測(cè)定病毒滴度。因此慢病毒轉(zhuǎn)染相對(duì)于常規(guī)質(zhì)粒轉(zhuǎn)染，技術(shù)難度更高，周期更長(zhǎng)。

RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.

5' LTR-ΔU3: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that in wildtype virus, the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript. On our vector, 5' LTR-ΔU3 is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the RSV promoter engineered upstream of 5'LTR-ΔU3 LTR.

Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.

RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.

cPPT: HIV-1 Central polypurine tract. It creates a "DNA flap" that increases nuclear import of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.

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

Lox2272: Recombination site for Cre recombinase. Mutated Lox site with two base substitutions of wild type LoxP. Incompatible with LoxP sites. When Cre is present, the LoxP and LoxP2272 sites will be cut and recombine with compatible sites.

LoxP: Recombination site for Cre recombinase. Incompatible with Lox2272 sites. When Cre is present, the LoxP and Lox2272 sites will be cut and recombine with compatible sites.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF #1: The open reading frame of a gene of interest is placed here, in a sense orientation. This gene can be expressed without Cre-mediated recombination.

ORF #2: The open reading frame of a gene of interest is placed here, in an antisense orientation. This gene can only be expressed after Cre-mediated recombination.

WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances transcriptional termination in the 3' LTR during viral RNA transcription, which leads to higher levels of functional viral RNA in packaging cells and hence greater viral titer. It also enhances transcriptional termination during the transcription of the user's gene of interest on the vector, leading to their higher expression levels.

mPGK promoter: Mouse phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

3' LTR-ΔU3: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (since the 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in 3' LTR-ΔU3 serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.

SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.