哺乳動物基因FLEX條件性表達慢病毒載體（Cre-Switc吃房h）

LEX條件性表達慢病毒載體結合了載體家的高效的第三代慢病毒載體系城對統和Cre響應的FLEX條件性基因表你路達載體，可以幫助您實現在多種(zhǒng)多樣(yàng)的哺乳動物樹愛細胞中以慢病毒轉導Cre響應的FLEX基因表達睡從調控開(kāi)關。FLEX Cre-Switch系統的兩(坐懂liǎng)個Lox-變體重組位點之間包票醫含一對(duì)反向(xiàng紙行)平行的ORF，在Cre重組酶的調這裡控下ORF上的兩(liǎng)個目的基因編碼方向(xiàng)反轉，從錢物而讓一個目的基因從激活表達變成(chéng)沉默吃下，另一個目的基因從沉默變成(c是頻héng)激活表達。

FLEX Cre-switch都老系統由兩(liǎng)對(duì)異型Lox-變體重見會組位點組成(chéng)，其中野生型序列稱爲LoxP，突變體稱藍長爲Lox2272，分别位于兩(liǎng)個基因ORF的兩(liǎng)側。習服 兩(liǎng)個ORF相互反向(xiàng)，其中雜吃一個ORF具有正确的讀碼方向(xiàng)（相對(duì)于啓動子）。兩學風(liǎng)種(zhǒng)Lox-變體都(dōu)可被(bèi視懂)Cre識别，但隻有相同的Lox位點對(duì)可以彼此重組的為，與其他Lox-變體不能(néng)進(jìn制說)行重組。LoxP和Lox2272位點以交替方式位如工于兩(liǎng)個ORF兩(liǎng)端，報南每對(duì)位點互爲反向(xiàng)吧坐。Cre重組酶不存在的情況時(shí)，第一個ORF由于其編愛年碼方向(xiàng)與啓動子方向(xiàng)相生裡同，可以在客戶定制的啓動子驅動又視下正常表達，而第二個ORF由于答要其方向(xiàng)相反則無法正常表達。在Cre的存員知在下，LoxP和Lox2272位點對(d樂遠uì)分别重組，導緻兩(liǎng)個ORF方向(xià如兵ng)反轉，且其中的一對(duì)重組位點將(銀就jiāng)具有相同的方向(xiàng)，Cre微校會(huì)介導産生正向(xiàng)重組切劇我割，繼而分别留下一個不同的重組位點。 ORF他雨方向(xiàng)的反轉，使得第二個OR可以在啓動子的驅動下正常表達，而第一線行個ORF將(jiāng)不表達。 由于ORF側少報翼是兩(liǎng)個不同的Lox-變體位點樂公，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粒的方式構建并使用E. c音學oli擴增，然後(hòu)與多個輔助質粒一同轉染至包裝細胞。載體上的兩(li他很ǎng)個LTR區域之間的DNA序上山列將(jiāng)被(bèi)轉錄成(chéng)RNA，而輔冷志助質粒表達的病毒蛋白進(jìn)一步與這(z低兒hè)些RNA組裝形成(chéng)完整的病毒顆粒。有活性的病毒顆粒被(bèi有身)釋放到上清液中。病毒上清液可直接轉染或者濃縮後(hòu)轉染細胞能友。

當慢病毒感染靶細胞時(shí)，病毒RNA被(bèi)反轉錄著妹成(chéng)DNA，然後(hòu)永久性整合到宿主基因組。位于兩(li的得ǎng)個LTR區域之間的載體組分永久整友文合到宿主基因組。對(duì)于上述的FLEX介導的Cre-Switch些錢條件性表達慢病毒載體，FLEX Cre-Switch調控的兩(liǎn民鄉g)個反向(xiàng)平行的ORF位于兩(得輛liǎng)個LTR之間的區域。這(zhè)兩(liǎn懂村g)個ORF也被(bèi)永久整合到宿主基因組當中，并在Cr秒得e調控下使得其中一個ORF獲得激活表達，另一個則表達失活。

關于該載體系統的更多信息，請參考以下文獻。

FLEX Cre-Switch條件性表達慢病毒載體專爲在舊兵哺乳動物細胞和動物體内實現兩(liǎng)個ORF可切換的什放基因表達而設計。目的基因表達受到用戶自定事兵義的啓動子調節，并在Cre重組酶共表達的情況下沉默一個目的基因，并激活另一就我個目的基因表達。

我們目前采用的是第三代慢病毒包裝載低你體系統。經(jīng)優化，該載體在大腸杆菌體内具有很高的拷貝數睡家，包裝的活病毒具有很高的滴度，對(duì)大多數宿主細胞具有下好高效的轉導能(néng)力，能著我(néng)有效地把載體整合到靶細胞基來在因組并實現外源基因的高水平表達。

載體容量受限：野生型的慢病毒基因組大小約爲9.2 kb，而在我們的慢病毒載體中，病毒包裝和金拍轉導的必要元件約爲2.8 kb是短，餘下6.4 kb的空間容納客戶的目的序列。當病毒載體超過(guò)以上大麗少小限制，病毒的包裝滴度將(jiāng)會(huì)大大降低。我們子腦的慢病毒載體除了可以插入靶基因的序列外，票廠還(hái)可以插入啓動子和篩選标記等載體元件。如果目的媽算基因和這(zhè)些載體元件長(c村嗎háng)度超過(guò)了6.4 kb，病毒的産船熱量有可能(néng)會(huì)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，然話新後(hòu)測定病毒滴度。因此慢病毒轉染相對(duì)于常規質粒轉染，技術難度厭請更高，周期更長(cháng)。

RSV promoter: Rous sarcoma virus pr子呢omoter. It drives transcri水哥ption of viral RNA in packa這窗ging cells. This RNA is then pa可還ckaged into live virus是房.

5' LTR-ΔU3: A deleted version of the HIV-1遠間 5' long terminal repeat. In花中 wildtype lentivirus子土, 5' LTR and 3' LTR少如 are essentially identical i老劇n sequence. They reside on two end金化s of the viral genome and po照對int in the same direction. Upon vi音這ral integration, the 3'做還 LTR sequence is cop理玩ied onto the 5' LTR. Th市少e LTRs carry both promoter and polya放光denylation function, such that in 為妹wildtype virus, the 5' LTR acts as a pr微女omoter to drive 空又the transcription of the viral 山長genome, while the 3' LTR acts as a poly草金adenylation signal to terminat跳慢e the upstream transcript.對兵 On our vector, 5' LTR-ΔU3 is 謝化deleted for a region 自女that is require多資d for the LTR's技購 promoter activity normally facilita制商ted by the viral tr路服anscription factor Tat. 化們This does not affect the production of火快 viral RNA during packaging beca現可use the promoter function is s花長upplemented by the RSV promoter engi身吧neered upstream of 5'LTR-ΔU3 LTR.

Ψ: HIV-1 packaging 費畫signal required for t做紙he packaging of viral RNA into vir站舊us.

RRE: HIV-1 Rev respons村文e element. It allows the n喝河uclear export of 音拍viral RNA by the viral Rev prot那有ein during viral packaging.

cPPT: HIV-1 Central polypurine tract. It 上用creates a "DNA flap" that increases nu學討clear import of the viral genome d章到uring target cell infe生短ction. This improves vector 身件integration into the host genome,地著 resulting in higher transduction eff森雨iciency.

Promoter: The promoter dr睡用iving your gene of int東是erest is placed here.

Lox2272: Recombination site for Cre議拍 recombinase. Mutat匠匠ed Lox site with 能熱two base substitutions of w知資ild type LoxP. Incompatible with LoxP 在但sites. When Cre is 化農present, the LoxP and LoxP2272 s工票ites will be cut and謝黃 recombine with co資都mpatible sites.

LoxP: Recombina窗紅tion site for Cre recombi和件nase. Incompatible with Lox2272 又現sites. When Cre雜光 is present, the LoxP and Lox呢道2272 sites will 間區be cut and recombin去窗e with compatible sites.

Kozak: Kozak consensus sequence話一. It is placed in front of 車好the start codon就費 of the ORF of interest bec熱如ause it is believed to facili睡可tate translation in人笑itiation in eukar拿拍yotes.

ORF #1: The open reading frame of a ge河跳ne of interest is placed here, 內來in a sense orientation. This ge線音ne can be expressed witho門遠ut Cre-mediated recombination.

ORF #2: The open read關視ing frame of a gene什玩 of interest is placed學站 here, in an antisense很這 orientation. This g工西ene can only be expressed after Cre-雜廠mediated recomb火通ination.

WPRE: Woodchuck h綠鐘epatitis virus pos外鐵ttranscriptional regulatory element. It著務 enhances transcriptional termination樂舞 in the 3' LTR during vir黑討al RNA transcrip時很tion, which leads to higher leve件報ls of functional viral RNA in packagin熱什g cells and hence greater viral titer吧聽. It also enhances tran理雜scriptional ter水去mination during t文金he transcription of the用自 user's gene of interest on the vecto又文r, leading to their higher 厭舊expression levels.

mPGK promoter: Mouse phosp個低hoglycerate kinase 1 gene promo不樂ter. It drives th間行e ubiquitous expre姐土ssion the downstream m坐看arker gene.

Marker: A drug selection gene (such as 微房neomycin resistance), 呢金a visually detectable gene (such 問花as EGFP), or a dual-reporter gene (亮湖such as EGFP/Neo). This a微和llows cells transduced 謝行with the vector to be selecte火頻d and/or visuali河水zed.

3' LTR-ΔU3: A truncated version of the 火對HIV-1 3' long terminal repeat tha的呢t deletes the U3 region. This lead術文s to the self-inactivati花那on of the promoter activity of醫也 the 5' LTR upon viral v我歌ector integration into the h場和ost genome (since the 3' LTR is copi答船ed onto 5' LTR du多著ring viral integration). The polya花讀denylation signal contained 拿店in 3' LTR-ΔU3 serves to 議志terminates all upstream t爸窗ranscripts produc唱員ed both during 生開viral packaging and after viral 嗎器integration into the host ge紙風nome.

SV40 early pA: Simian virus 40 early polya書姐denylation signal. It further faci但劇litates transcri區白ptional termination after the 人都3' LTR during viral 就舞RNA transcripti有報on during packaging. 東票This elevates t近東he level of functional viral他日 RNA in packaging cell看舞s, thus improving viral titer.

Ampicillin: Ampicillin resistance g黃師ene. It allows t呢為he plasmid to be maintained市的 by ampicillin selection in E. 兵能coli.

pUC ori: pUC origin of replication這體. Plasmids carrying this 票長origin exist in high copy nu遠事mbers in E. coli.

基因激活失活可控：兩(liǎng)個ORF的方向(xiàng)彼此相反友拍确保當表達正向(xiàng)方向(xiàng)的ORF時(sh關開í)，反向(xiàng)的ORF被(bèi)抑制，可防止基因發(fā)生洩漏哥我表達。

外源基因的穩定整合：常規質粒轉染隻能(néng)實現外源基因的瞬時(shí)表達，這(zhè)種腦習(zhǒng)外源基因會(huì)随著(zhe)宿主媽生細胞的分裂而不斷丢失，在快速分裂的細胞中顯得尤爲顯著。相反的是，慢內吧病毒轉導的目的基因能(néng)穩定地整合到宿主細胞的染色從年體中 ，因而會(huì)随著(zhe)宿主細喝是胞的分裂而穩定遺傳。

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算費服務，病毒滴度可以達到>10⁹ TU/ml。在這(zhè)樣(yàng)的病毒滴度下，很外如果選擇合适的劑量去轉導體外培養的哺乳動物細胞，則轉導效率可接習照近100%。

宿主範圍廣泛：我們的病毒包裝系統包裝出來的病毒含有VSV-錢是G包膜蛋白，此蛋白擁有非常廣泛的親和性，可以轉導能廠幾乎所有的哺乳動物細胞，包括分裂細胞，非分裂細胞，原代細胞公一，穩定細胞系，幹細胞，分化細胞，貼壁細胞和懸浮細胞等各類哺乳動物細花明胞，甚至還(hái)可以轉導一些非城美哺乳動物細胞。使用傳統的轉染方式轉導神經(jīng)元細胞是非醫村常難的，但是采用我們慢病毒載體系統可以輕易的實現神用事經(jīng)元細胞的轉導。相對(duì)城外于在某些細胞中具有較低轉導效率的腺病毒和不能(néng)用于非分裂草還細胞的逆轉錄病毒而言，利用我們的慢病毒包裝系統包裝出來小地的病毒具有廣泛的親和性。

基因拷貝數相對(duì)均一：通常情況下，采用病毒轉導的方式可以比較均一黑為的將(jiāng)外源基因轉入靶細胞中，而傳統的質粒轉影船染則呈現出較高的不均一性，導緻某些細胞會(山吃huì)獲得較多拷貝質粒而某些則會(huì)獲得較少甚至完全沒(méi山亮)有。

體内外實驗均有效：我們的載體不僅擁有良好(hǎo)的體外細胞轉導能(néng)力，同樣(y歌小àng)适用于體内活體動物實驗。

安全性高：我們的病毒載體系統具備了以下兩(liǎng)靜友大特點，因而具有非常高的安全性。一、病毒包裝和轉導所必需的基因由三個輔助質知那粒分開(kāi)表達。二、5' 舊秒LTR的啓動子自失活。因此，在進(jì月裡n)行病毒包裝和病毒轉導的時(shí)候不快資會(huì)産生具有複制能(néng)力的師技病毒顆粒，使用我們的載體對(d美小uì)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爲9.2 kb，而在我們的慢病電什毒載體中，病毒包裝和轉導的必要元件約爲那月2.8 kb，餘下6.4 kb的空間容納客戶的目的序列。當病毒載體超過(guò跳離)以上大小限制，病毒的包裝滴度將(jiāng)短紙會(huì)大大降低。我們的慢病毒載多鄉體除了可以插入靶基因的序列外，還(hái)可以插入啓動子和篩選标記等載體元件。制體如果目的基因和這(zhè)些載體元件長(cháng)度超過(guò)笑都了6.4 kb，病毒的産量有可能(nén業草g)會(huì)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友訊，然後(hòu)測定病毒滴度。因此睡匠慢病毒轉染相對(duì)于常規質粒轉染，技術難度更高，周期更吃視長(cháng)。

RSV promoter: Rous sarcoma virus錢務 promoter. It drives transcription愛刀 of viral RNA in packaging cells. 白有This RNA is then packaged in能志to live virus.

5' LTR-ΔU3: A deleted version of the HIV-1 5' lon對水g terminal repeat. In 紅去wildtype lentivir男離us, 5' LTR and 3' LTR are essentiall醫南y identical in 新錢sequence. They resid呢資e on two ends of the viral genome an一厭d point in the same dir草這ection. Upon viral i我笑ntegration, the 3刀北' LTR sequence is copied onto 銀藍the 5' LTR. The LTRs carr公工y both promoter and polyadenyl制農ation function, such that in wil高街dtype virus, the 5' LTR acts as a pr身匠omoter to drive the transcr電冷iption of the viral genome, 道著while the 3' LTR acts as a訊市 polyadenylation signal to termin林低ate the upstream tran在腦script. On our vecto子樂r, 5' LTR-ΔU3 is deleted for a現作 region that is required for the LTR'可日s promoter activity normally fa空票cilitated by the viral transc新術ription factor Tat. This do呢冷es not affect the produc湖身tion of viral RNA during packa媽作ging because the promoter function is校草 supplemented by the 件下RSV promoter engineered upstream of 5樹草'LTR-ΔU3 LTR.

Ψ: HIV-1 packaging signal required fo長我r the packaging of 姐劇viral RNA into virus.信刀

RRE: HIV-1 Rev response element. It低白 allows the nuc為站lear export of viral R低市NA by the viral R可內ev protein during viral packa計子ging.

cPPT: HIV-1 Central polypurine tract. It cre謝喝ates a "DNA flap" tha腦老t increases nuclear import of th輛錢e viral genome durin去喝g target cell inf音器ection. This improves vector in很他tegration into the host報下 genome, resulting in hig也可her transduction efficienc來低y.

Promoter: The promoter driving 麗業your gene of interest is plac白他ed here.

Lox2272: Recombina議通tion site for Cre rec年多ombinase. Mutated Lox site with two 低醫base substitutions of報信 wild type LoxP. Incompatible with 懂什LoxP sites. When Cre is present,站區 the LoxP and LoxP2272 sites制議 will be cut and recombine with com地湖patible sites.

LoxP: Recombination site for Cre recom件土binase. Incompatib友放le with Lox2272 sites. Whe男湖n Cre is present, t志畫he LoxP and Lox2272 sites will be 裡她cut and recombine with compatible水樹 sites.

Kozak: Kozak consensus sequ劇些ence. It is placed in fron妹亮t of the start codon of the ORF of場資 interest because i我亮t is believed to facilita鄉地te translation init藍吧iation in eukaryot訊黑es.

ORF #1: The open read街師ing frame of a gene of interest筆河 is placed here, in a se男路nse orientation. This gene can be e行個xpressed without Cre-m呢多ediated recombination.

ORF #2: The open r那你eading frame of a gene of in城腦terest is placed here,林火 in an antisense o業術rientation. This ge和場ne can only be exp那暗ressed after Cre-mediated recombin訊刀ation.

WPRE: Woodchuck hepatitis viru黃火s posttranscriptional regulatory 我請element. It enhances transcri黃光ptional termination in t可又he 3' LTR during vir低空al RNA transcription,信站 which leads to h通可igher levels of funct厭什ional viral RNA i下姐n packaging cells and hence greater腦紙 viral titer. It also enha金微nces transcriptional t多兒ermination during the和紙 transcription of 音區the user's gene of inter筆麗est on the vector, leading to 花問their higher expression levels.

mPGK promoter: Mouse phosphoglycerate k月歌inase 1 gene promoter. It d來上rives the ubiquitous express司匠ion the downstr購和eam marker gene.

Marker: A drug selection gene (such as 不短neomycin resistance), a visually detec紙個table gene (suc器鐘h as EGFP), or a dual-reporter gene (su厭些ch as EGFP/Neo). This allows cells t新費ransduced with the vector 明如to be selected and/or visualized.

3' LTR-ΔU3: A truncated version of th身東e HIV-1 3' long terminal repeat t技票hat deletes the U3 re窗雜gion. This lead事上s to the self-inacti街長vation of the promoter activit服國y of the 5' LTR upon又紅 viral vector integ這風ration into the host gen人們ome (since the 3' LTR is明現 copied onto 5' LTR during 生高viral integration). The po聽美lyadenylation sig月外nal contained in 3' LTR-ΔU3 se明月rves to terminates all 坐劇upstream transcripts produced both吧線 during viral pac線近kaging and after viral integratio校短n into the host文說 genome.

SV40 early pA: Simian virus 40 early窗答 polyadenylation signal. 店明It further facilitates transcriptional 樂很termination after the 3'慢拿 LTR during viral RNA transcript男來ion during packaging. This el靜放evates the level行藍 of functional viral RNA 他我in packaging cells, th裡購us improving viral 廠女titer.

Ampicillin: Ampicillin會做 resistance gene. It allows the老匠 plasmid to be maintained b拿金y ampicillin select文短ion in E. coli.

pUC ori: pUC origin of replication. Plasmids ca文老rrying this origin ex身器ist in high copy numb地高ers in E. coli.