慢病毒整合穩(wěn)定細(xì)胞株
▏ 名詞定義
慢病毒：慢病毒是一種逆轉(zhuǎn)錄病毒，屬于逆轉(zhuǎn)錄病毒科。慢病毒表達(dá)逆轉(zhuǎn)錄酶和整合酶，前者將病毒RNA轉(zhuǎn)化為雙鏈DNA，后者將病毒DNA插入宿主DNA，會(huì)隨宿主細(xì)胞一起分裂。這一特性可用于將各種基因、突變或治療藥物穩(wěn)定地遞送到細(xì)胞中，幾十年來(lái)已在研究界得到廣泛應(yīng)用。一般認(rèn)為，逆轉(zhuǎn)錄病毒只感染分裂細(xì)胞，而慢病毒則同時(shí)感染分裂細(xì)胞和非分裂細(xì)胞。
野生型慢病毒已被改造成可在實(shí)驗(yàn)室環(huán)境中安全使用的慢病毒載體。這些改造的慢病毒載體具有諸多優(yōu)勢(shì)，使其成為多種細(xì)胞類型和模型中有用的研究工具。慢病毒載體可以靶向非分裂細(xì)胞，能夠在宿主細(xì)胞中長(zhǎng)期穩(wěn)定表達(dá)，并且具有較低的免疫原性。
慢病毒載體： 慢病毒的完整基因組通常8-10kb，由單鏈RNA編碼，包含諸多元件，但是實(shí)驗(yàn)用于包裝的慢病毒，出于安全性的考慮，許多元件都被突變、移除。
圖1 慢病毒的完整基因組元件圖示
1. 包裝基因包含：gag、pol、env
2. 調(diào)控基因包含：tat、rev
3. 輔助基因包含：vif、vpr、vpu、nef
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Transfer plasmid |
LTR |
in cis |
Long terminal repeat; comprised of a U3-R-U5 structure and are found on each side of the provirus. The U3 (unique 3’) contains sequences necessary for activation of viral genomic RNA transcription. R is the repeat region. |
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U3 |
in cis |
Unique 3’; contains sequences necessary for activation of viral genomic RNA transcription. Removal of this region in the 3’ LTR in third-generation plasmids creates self-inactivating viral vectors. |
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R |
in cis |
Repeat region where Tat binds. |
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TAR |
in cis |
Trans-activating response element; found in the R region and acts as the binding site for Tat. |
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U5 |
in cis |
Unique 5'; in third-generation plasmids, this region is often removed in 5’ LTRs and replaced with a heterologous promoter (CMV or RSV). |
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5' LTR |
in cis |
Acts as an RNA pol II promoter; the transcript begins, by definition, at the beginning of R, is capped, and proceeds through U5 and the rest of the provirus. Third-generation plasmids use a hybrid 5' LTR with a constitutive promoter such as CMV or RSV. |
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3' LTR |
in cis |
Terminates transcription started by 5' LTR by the addition of a polyA tract just after the R sequence. |
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WPRE |
in cis |
Woodchuck hepatitis virus post‐transcriptional regulatory element; stimulates the expression of transgenes via increased nuclear export. |
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RRE |
in cis |
Rev Response Element; he sequence to which the Rev protein binds. |
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Psi (?) |
in cis |
RNA packaging signal; recognized by nucleocapsid proteins and essential for efficient viral packaging. |
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cPPT |
in cis |
Central polypurine tract; recognition site for proviral DNA synthesis. Increases transduction efficiency and transgene expression. |
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Packaging plasmid |
gag |
in trans |
Precursor structural protein of the lentiviral particle containing matrix, capsid, and nucleocapsid components. |
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pol |
in trans |
Precursor protein containing reverse transcriptase and integrase components. |
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rev |
in trans |
Binds to the Rev Response Element (RRE) within unspliced and partially spliced transcripts to facilitate nuclear export. Provided by a separate plasmid from gag/pol in third-generation packaging plasmids. |
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tat |
in trans |
Trans-activator; binds TAR to activate transcription from the LTR promoter. Only in first- and second-generation lentiviral plasmids. |
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Envelope plasmid |
env |
in trans |
The viral envelope gene; typically vesicular stomatitis virus G glycoprotein (VSV-G), an envelope protein with broad tropism used to pseudotype most lentiviral vectors. |
