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首頁(yè) ? Flp-in system穩(wěn)定表達(dá)細(xì)胞株構(gòu)建方法- FLP重組酶介導(dǎo)的基因整合

Flp-in system穩(wěn)定表達(dá)細(xì)胞株構(gòu)建方法- FLP重組酶介導(dǎo)的基因整合

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Flp-in system               Vectors:pFRT/lacZeo, pcDNA5/FRT, pcDNA5/FRT/TO, pOG44

    研究一個(gè)基因的功能，最常規(guī)的方法便是過(guò)表達(dá)（over-expression）和靜默（knock-down或者knock-out），一般是通過(guò)外源基因轉(zhuǎn)染宿主，進(jìn)而觀察宿主相關(guān)的表型（Phenotype）發(fā)生的變化（凋亡、增殖、周期、侵襲、遷移、上下游信號(hào)通路、EMT等等）來(lái)判斷基因的功能。
    不管是過(guò)表達(dá)還是靜默，細(xì)胞水平的研究往往是選擇構(gòu)建穩(wěn)定細(xì)胞株，研究細(xì)胞發(fā)生的變化。常規(guī)的構(gòu)建穩(wěn)定細(xì)胞株的方法有真核質(zhì)粒轉(zhuǎn)染、慢病毒包裝。慢病毒包裝周期較長(zhǎng)，費(fèi)用較高，對(duì)實(shí)驗(yàn)室硬件條件要求高，極大地限制了他的應(yīng)用；普通的真核質(zhì)粒轉(zhuǎn)染周期較長(zhǎng)，目的基因整合隨機(jī)，對(duì)宿主要求高等缺點(diǎn)也限制其應(yīng)用。隨著新技術(shù)的不斷革新，針對(duì)真核質(zhì)粒轉(zhuǎn)染構(gòu)建穩(wěn)定細(xì)胞株的方法也不斷的創(chuàng)新，出現(xiàn)TALEN、IOS、Cas9、Flp-In等技術(shù)，不斷地提高穩(wěn)定細(xì)胞株構(gòu)建的成功率。
    尤其是Flp-In系統(tǒng)，以其獨(dú)特的優(yōu)點(diǎn)，迅速被各大實(shí)驗(yàn)室采用，近些年累計(jì)發(fā)表的文獻(xiàn)越來(lái)越多，得到廣大科研工作者的認(rèn)可。



Flp-In? System
For Generating Stable Mammalian Expression Cell Lines by Flp Recombinase-Mediated Integration


Flp-In? System總覽：
Flp-In?完整系統(tǒng)(Flp-In? Complete System)可使目的基因在哺乳動(dòng)物細(xì)胞基因組的特定位點(diǎn)整合和表達(dá)。Flp-In系統(tǒng)(Flp-In System)涉及將Flp重組靶(FRT)位點(diǎn)導(dǎo)入所選哺乳動(dòng)物細(xì)胞系的基因組中。然后通過(guò)Flp重組酶介導(dǎo)在FRT位點(diǎn)的DNA重組，將含有目的基因的表達(dá)載體整合到基因組中。
Flp-In?系統(tǒng)(Flp-In? System)的主要組分包括：
1. Flp-In靶位點(diǎn)載體pFRT?lacZeo，用于制備含有FRT整合位點(diǎn)的宿主細(xì)胞系
2. 含有與潮霉素抗性基因相連的FRT位點(diǎn)的表達(dá)質(zhì)粒，用于在Flp重組酶介導(dǎo)下，整合和篩選表達(dá)目的基因的穩(wěn)定細(xì)胞系；目的基因的表達(dá)由人巨細(xì)胞病毒(CMV)即刻早期增強(qiáng)子?啟動(dòng)子調(diào)控
3. Flp重組酶表達(dá)質(zhì)粒pOG44，用于在人CMV啟動(dòng)子調(diào)控下表達(dá)Flp重組酶
4. 含有氯霉素乙?；D(zhuǎn)移酶(CAT)基因的對(duì)照表達(dá)質(zhì)粒，在與pOG44共轉(zhuǎn)染到Flp-In宿主細(xì)胞系中時(shí)表達(dá)CAT

Flp-In? System的優(yōu)點(diǎn)：
1. 一旦成功構(gòu)建含F(xiàn)RT整合位點(diǎn)的Flp-In母細(xì)胞株，接下來(lái)構(gòu)建表達(dá)目的基因穩(wěn)定細(xì)胞株的工作就快速、高效；
2. Flp-In系統(tǒng)能構(gòu)建同基因型的穩(wěn)定細(xì)胞株；
3. Flp-In系統(tǒng)可以是多克隆穩(wěn)定細(xì)胞株，無(wú)需純化。  

Flp-In? System描述：
Flp-In系統(tǒng)依據(jù)釀酒酵母的DNA重組系統(tǒng)的特點(diǎn)，高效構(gòu)建穩(wěn)定哺乳動(dòng)物表達(dá)細(xì)胞株。這種DNA重組系統(tǒng)應(yīng)用重組酶（Flp）和定點(diǎn)重組技術(shù)(Craig, 1988; Sauer, 1994)，將目的基因插入到哺乳動(dòng)物指定的基因組中。
Flp-In系統(tǒng)運(yùn)用3種不同的質(zhì)粒來(lái)構(gòu)建同基因型的穩(wěn)定細(xì)胞株。
pFRT/lacZeo質(zhì)粒是用來(lái)構(gòu)建Flp-In母細(xì)胞株。質(zhì)粒包含lacZ-Zeocin融合基因，有SV40早期啟動(dòng)子控制表達(dá)。FRT位點(diǎn)被插入在lacZ-Zeocin融合基因的ATG起始密碼子下游。FRT位點(diǎn)是用來(lái)與Flp重組酶結(jié)合，進(jìn)而被剪切。pFRT/lacZeo轉(zhuǎn)染進(jìn)細(xì)胞中，然后通過(guò)Zeocin抗生素篩選細(xì)胞，陽(yáng)性克隆細(xì)胞即含單一的FRT整合位點(diǎn)。Flp-In母細(xì)胞株含F(xiàn)RT位點(diǎn)和表達(dá)lacZ-Zeocin融合基因。pFRT/lacZeo質(zhì)粒整合進(jìn)入基因組是隨機(jī)的。
第二個(gè)主要的質(zhì)粒是pcDNA5/FRT表達(dá)載體，用來(lái)將目的基因克隆進(jìn)去，目的基因由hCMV啟動(dòng)子控制，載體還有Hygromycin抗體基因，還有5’編碼區(qū)的FRT位點(diǎn)。Hygromycin抗性基因缺少啟動(dòng)子和ATG起始密碼子。
第三個(gè)主要的質(zhì)粒是pOG44載體，用來(lái)表達(dá)Flp重組酶(Broach et al., 1982; Broach and Hicks, 1980; Buchholz et al., 1996)，由hCMV啟動(dòng)子控制。
pOG44質(zhì)粒和含目的基因的pcDNA5/FRT質(zhì)粒共轉(zhuǎn)Flp-In母細(xì)胞株，F(xiàn)lp重組酶介導(dǎo)FRT位點(diǎn)的同源重組（母細(xì)胞基因組和pcDNA5/FRT），這樣pcDNA5/FRT的目的基因插入基因組。同時(shí)將Hygromycin抗性基因插入到pFRT/lacZeo的SV40啟動(dòng)子和ATG起始密碼子下，抑制了lacZ-Zeocin融合基因的表達(dá)。這樣的話Flp-In穩(wěn)定細(xì)胞株就可以通過(guò)hygromycin耐受、Zeoncin敏感、缺少?-半乳糖苷酶活性、表達(dá)目的基因4個(gè)特性去篩選得到。

Flp-In? System流程圖：
下圖描述了Flp-In系統(tǒng)的主要流程：
flp-in系統(tǒng)流程圖

Flp重組酶介導(dǎo)DNA的重組：
在Flp-In系統(tǒng)中，F(xiàn)lp重組酶介導(dǎo)分子間的DNA重組，F(xiàn)lp重組酶介導(dǎo)的重組有如下特點(diǎn)：
1. 重組發(fā)生在特異的FRT位點(diǎn)；
2. 重組很保守，不需要DNA合成，F(xiàn)RT重組位點(diǎn)被保護(hù)，使重組位點(diǎn)發(fā)生突變的可能性降到最低；
3. 重組僅僅需要34bpFRT位點(diǎn)。
更多關(guān)于Flp重組酶和保守位點(diǎn)特異重組請(qǐng)參考文獻(xiàn)(Craig, 1988; Sauer, 1994).

FRT位點(diǎn)：
FRT位點(diǎn)最初從釀酒酵母中分離得到，并被深入研究(Gronostajski and Sadowski, 1985; Jayaram, 1985; Sauer, 1994; Senecoff et al., 1985).最短的FRT位點(diǎn)包含34bp序列，包含2個(gè)13bp的片段序列，中間8bp序列含Xba I限制性酶切位點(diǎn)，另外13bp重復(fù)序列在大多數(shù)FRT位點(diǎn)中也有，但是并不是剪切所必須的(Andrews et al., 1985).當(dāng)Flp重組酶結(jié)合到3段13bp的序列上時(shí)，剪切發(fā)生在中間的8bp區(qū)域(Andrews et al., 1985; Senecoff et al., 1985).
序列

實(shí)驗(yàn)流程：

  1. 將pFRT/lacZeo質(zhì)粒轉(zhuǎn)染細(xì)胞，構(gòu)建Flp-In母細(xì)胞株；

  2. 將目的基因克隆進(jìn)pcDNA5/FRT表達(dá)載體；

  3. 在Flp-In母細(xì)胞中共轉(zhuǎn)pcDNA5/FRT和pOG44質(zhì)粒，構(gòu)建出Flp-In表達(dá)細(xì)胞株；

  4. 檢測(cè)目的基因的表達(dá)。


flp-in系統(tǒng)流程圖

Flp-In?母細(xì)胞株構(gòu)建：
BioVector NTCC Inc.從Invitrogen公司購(gòu)得Flp-In?-293、Flp-In?-CV-1、Flp-In?-CHO、Flp-In?-BHK、Flp-In?-3T3、Flp-In?-Jurkat，節(jié)約客戶的時(shí)間，同時(shí)也提供其他細(xì)胞上的的Flp-In母細(xì)胞株構(gòu)建服務(wù)。

Cell Line

Source

Catalog no.

Flp-In?-293

Human embryonic kidney

NTCC600101

Flp-In?-CV-1

African Green Monkey kidney

NTCC600102

Flp-In?-CHO

Chinese Hamster ovary

NTCC600103

Flp-In?-BHK

Baby hamster kidney

NTCC600104

Flp-In?-3T3

Mouse (NIH Swiss) embryonic fibroblast

NTCC600105

Flp-In?-Jurkat

Human T-cell leukemia

NTCC600106

案例展示：

Figure 4 Cell migration and proliferation assay of TMEM16A variants.A, Representative images of wound healing in a scratch assay with inducible expression of TMEM16A variants in HEK293 cells cultured with (Tet+) or without (Tet?) tetracycline (0.1 μg/ml). Original magnification, 4x. (Scale bars: 5 μm). B, Quantification of the fraction of the wound that remains uncovered by the migratory cells as a function of time for cell treated with (Tet+) or without (Tet?). C, Cellular proliferation assay, BrdU staining of cells expressing TMEM16A variants (treated without or with tetracycline). Data represent the % of BrdU+ cells and are the mean ± SD of three independent experiments.

All the coding sequences for TMEM16A were cloned in the pcDNA5 FRT/TO plasmid. Stable expression of TMEM16A variants was achieved by Flp-recombinase-mediated recombination in HEK293 Flp-In cells followed by hygromycin B selection. Each TMEM16A-expressing vector that expresses the Flp-recombinase was cotransfected with Effectene transfection reagent and selected with a concentration of 200μg/ml hygromycin B. Individual clones were obtained by limited dilution. Induction of TMEM16A isoforms expression was achieved with 0.1μg/ml tetracycline . Cells were grown in DMEM-Glutamax-I media supplemented with 5% fetal bovine serum.

標(biāo)題：

TMEM16A alternative splicing coordination in breast cancer

雜志：Molecular Cancer
作者：

Ifeoma Ubby, Erica Bussani, Antonio Colonna, Giuseppe Stacul, Martina Locatelli, Paolo Scudieri, Luis Galietta and Franco Pagani


Figure 3. Inducible expression of hSLCO5A1 in HeLa cells. Protein expression of the YFP-tagged WT SLCO5A1 or its L33F mutant after induction with 1 mg/ml tetracycline for 24 h was analyzed by confocal fluorescence microscopy (blue: DAPI; yellow: YFP). The diagrams represent YFP fluorescence intensities along the length of the red arrows (x-axis: distance [μm]; y-axis: relative signal intensity).

Flp-In T-REx-HeLa cells allow the tetracycline-inducible expression of a gene of interest from a specific genomic location. Stable SLCO5A1-expressing Flp-In TREx-HeLa cells were generated using the FlpIn recombinasemediated system kit, which permits the targeted integration of genes to the same locus in all transfected cells to provide a homogeneous level of gene expression. To this end, cells were co-transfected with the FlpIn expression vector pcDNA5/FRT/TO (mock) or with the same vector containing the wild-type (WT) or mutant (L33F) sequence for SLCO5A1, modified C-terminally with either the sequence for a HA epitope or a YFP-tag or left unmodified, together with the Flp-recombinase expression vector pOG44. Individual clones were separated by monoclonal selection with 15 mg/ml blasticidin and 100 mg/ml hygromycinB. Cells were cultured in EMEM supplemented with 10% FCS (tetracycline/doxycycline-reduced). SLCO5A1-expression was induced by adding 1 mg/ml tetracycline (tet) to the Flp-In TREx-HeLa cells (hereinafter referred to as HeLa cells).

標(biāo)題：

Characterization of SLCO5A1/OATP5A1, a Solute Carrier Transport Protein with Non-Classical Function

雜志：

PLOS ONE

作者：

Katrin Sebastian, Silvia Detro-Dassen, Natalie Rinis, Dirk Fahrenkamp, Gerhard Mu¨ller-Newen, Hans F. Merk, Gu¨ nther Schmalzing, Gabriele Zwadlo-Klarwasser, Jens Malte Baron


Fig.1. PEX14–TEV–Protein A localizes to peroxisomes and is　functional. Validation of the peroxisomal localization of the Protein-A-tagged genomic copy of PEX14. Cell lines Flp-In-293 cells and Flp-In-293 [PEX14–TEV– Protein A] were transiently transfected with a plasmid encoding the peroxisomal marker EGFP–PTS1. Specific immunodetection of the fusion protein PEX14–TEV–Protein A was carried out with anti-Protein A antibodies, the endogenous and the fusion protein were detected by using antibodies against PEX14. Scale bars:10 um.

Generation and selection of stable cell lines expressing PEX14–TEV–ProteinA
Flp-In system was used for the generation of a stablePEX14–TEV–Protein A expression cell line Flp-In-293[PEX14–TEV–PA]. Flp-In-293 cells? were co-transfected with expression vector pPEX14–TEV–PA and the FLP-recombinase vector (pOG44) resulting in a stable integration of the gene of interest at the ‘FLP Recombination Target’ (FRT)-site in the genome. For the selective growth test, individual cell colonies were grown in duplicate on six-well plates and exposed to a confluence of 25%. The culture medium was supplemented with hygromycin (200 ug /ml) or Zeocin (100 ug/ml). The growth of possible integrants and Flp-In-293 cells as a control was monitored over a period of 7 days. Hygromycin-resistant and Zeocin-sensitive cells were further assayed for the lack of b-galactosidase activity. The expression of PEX14–TEV–ProteinA was verified by immunoblot analysis using anti-PEX14 antiserum.

標(biāo)題：

PEX14 is required for microtubule-based peroxisome motility in human cells

雜志：

Journal of Cell Science

作者：

Pratima Bharti, Wolfgang Schliebs, Tanja Schievelbusch, Alexander Neuhaus, Christine David, Klaus Kock, Christian Herrmann, Helmut E. Meyer, Sebastian Wiese, Bettina Warscheid, Carsten Theissand Ralf Erdmann


Figure 3. Confocal microscopy analysis and cell surface expression of Flp-In?-293 cells stablyexpressing wild-type and mutant BCRP. A) The cellular localization of wild-type and mutant BCRP in Flp-In?-293 cells (shown ingreen) was determined by immunofluorescent confocal microscopy using the BCRP-specific mAb BXP-21. Cell nuclei were stained with DAPI and are shown in red. B) Expression of wild-type and mutant BCRP on cell surface of stably transfected Flp-In?-293 cells was detected using the 5D3 monoclonal antibody. Representative flow cytometry histograms
showing cell surface expression of wild-type and mutant BCRP are presented. The red and blue peaks represent the phycoerythrin fluorescence associated with cells treated with the IgG2b negative control and the 5D3 antibodies, respectively. No surface expression of BCRP was detected in the pcDNA5 vector control cells.

Generation of Flp-In?-293 cells stably expressing wild-type BCRP and proline mutants
One μg of the pcDNA5/FRT plasmid containing wild-type or mutant BCRP cDNA or the empty vector were transfected into Flp-In?-293 cells in combination with 2 μg of pOG44 using the FuGENE HD transfection, according to the manufacturer’s instruction. After recovery, cells were selected in 125 μg/ml of hygromycin for approximately 3 – 4 weeks. The cells expressing wild-type or mutant BCRP were then maintained in complete DMEM medium containing 10% FBS, L-glutamine, and 125 μg/ml of hygromycin for subsequent experiments.

標(biāo)題：

Identification of Proline Residues In or Near the Transmembrane Helices of the Human Breast Cancer Resistance Protein (BCRP/ABCG2) Important for Transport Activity and Substrate Specificity

雜志：

Biochemistry

作者：

Zhanglin Ni, Zsolt Bikadi, Diana L. Shuster, Chunsheng Zhao, Mark F. Rosenberg, and Qingcheng Mao



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