Flp-in system穩定表達細胞株構建方法- FLP重組酶介導的基因整合

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

研究一個基因的功能，最常規的方法便是過表達（over-expression）和靜默（knock-down或者knock-out），一般是通過外源基因轉染宿主，進而觀察宿主相關的表型（Phenotype）發生的變化（凋亡、增殖、周期、侵襲、遷移、上下游信號通路、EMT等等）來判斷基因的功能。
不管是過表達還是靜默，細胞水平的研究往往是選擇構建穩定細胞株，研究細胞發生的變化。常規的構建穩定細胞株的方法有真核質粒轉染、慢病毒包裝。慢病毒包裝周期較長，費用較高，對實驗室硬件條件要求高，極大地限制了他的應用；普通的真核質粒轉染周期較長，目的基因整合隨機，對宿主要求高等缺點也限制其應用。隨著新技術的不斷革新，針對真核質粒轉染構建穩定細胞株的方法也不斷的創新，出現TALEN、IOS、Cas9、Flp-In等技術，不斷地提高穩定細胞株構建的成功率。
尤其是Flp-In系統，以其獨特的優點，迅速被各大實驗室采用，近些年累計發表的文獻越來越多，得到廣大科研工作者的認可。

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(Flp-In? Complete System)可使目的基因在哺乳動物細胞基因組的特定位點整合和表達。Flp-In系統(Flp-In System)涉及將Flp重組靶(FRT)位點導入所選哺乳動物細胞系的基因組中。然后通過Flp重組酶介導在FRT位點的DNA重組，將含有目的基因的表達載體整合到基因組中。
Flp-In?系統(Flp-In? System)的主要組分包括：
1. Flp-In靶位點載體pFRT?lacZeo，用于制備含有FRT整合位點的宿主細胞系
2. 含有與潮霉素抗性基因相連的FRT位點的表達質粒，用于在Flp重組酶介導下，整合和篩選表達目的基因的穩定細胞系；目的基因的表達由人巨細胞病毒(CMV)即刻早期增強子?啟動子調控
3. Flp重組酶表達質粒pOG44，用于在人CMV啟動子調控下表達Flp重組酶
4. 含有氯霉素乙酰基轉移酶(CAT)基因的對照表達質粒，在與pOG44共轉染到Flp-In宿主細胞系中時表達CAT

Flp-In? System的優點：
1. 一旦成功構建含FRT整合位點的Flp-In母細胞株，接下來構建表達目的基因穩定細胞株的工作就快速、高效；
2. Flp-In系統能構建同基因型的穩定細胞株；
3. Flp-In系統可以是多克隆穩定細胞株，無需純化。

Flp-In? System描述：
Flp-In系統依據釀酒酵母的DNA重組系統的特點，高效構建穩定哺乳動物表達細胞株。這種DNA重組系統應用重組酶（Flp）和定點重組技術(Craig, 1988; Sauer, 1994)，將目的基因插入到哺乳動物指定的基因組中。
Flp-In系統運用3種不同的質粒來構建同基因型的穩定細胞株。
pFRT/lacZeo質粒是用來構建Flp-In母細胞株。質粒包含lacZ-Zeocin融合基因，有SV40早期啟動子控制表達。FRT位點被插入在lacZ-Zeocin融合基因的ATG起始密碼子下游。FRT位點是用來與Flp重組酶結合，進而被剪切。pFRT/lacZeo轉染進細胞中，然后通過Zeocin抗生素篩選細胞，陽性克隆細胞即含單一的FRT整合位點。Flp-In母細胞株含FRT位點和表達lacZ-Zeocin融合基因。pFRT/lacZeo質粒整合進入基因組是隨機的。
第二個主要的質粒是pcDNA5/FRT表達載體，用來將目的基因克隆進去，目的基因由hCMV啟動子控制，載體還有Hygromycin抗體基因，還有5’編碼區的FRT位點。Hygromycin抗性基因缺少啟動子和ATG起始密碼子。
第三個主要的質粒是pOG44載體，用來表達Flp重組酶(Broach et al., 1982; Broach and Hicks, 1980; Buchholz et al., 1996)，由hCMV啟動子控制。
pOG44質粒和含目的基因的pcDNA5/FRT質粒共轉Flp-In母細胞株，Flp重組酶介導FRT位點的同源重組（母細胞基因組和pcDNA5/FRT），這樣pcDNA5/FRT的目的基因插入基因組。同時將Hygromycin抗性基因插入到pFRT/lacZeo的SV40啟動子和ATG起始密碼子下，抑制了lacZ-Zeocin融合基因的表達。這樣的話Flp-In穩定細胞株就可以通過hygromycin耐受、Zeoncin敏感、缺少?-半乳糖苷酶活性、表達目的基因4個特性去篩選得到。

Flp-In? System流程圖：
下圖描述了Flp-In系統的主要流程：
flp-in系統流程圖

Flp重組酶介導DNA的重組：
在Flp-In系統中，Flp重組酶介導分子間的DNA重組，Flp重組酶介導的重組有如下特點：
1. 重組發生在特異的FRT位點；
2. 重組很保守，不需要DNA合成，FRT重組位點被保護，使重組位點發生突變的可能性降到最低；
3. 重組僅僅需要34bpFRT位點。
更多關于Flp重組酶和保守位點特異重組請參考文獻(Craig, 1988; Sauer, 1994).

FRT位點：
FRT位點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位點包含34bp序列，包含2個13bp的片段序列，中間8bp序列含Xba I限制性酶切位點，另外13bp重復序列在大多數FRT位點中也有，但是并不是剪切所必須的(Andrews et al., 1985).當Flp重組酶結合到3段13bp的序列上時，剪切發生在中間的8bp區域(Andrews et al., 1985; Senecoff et al., 1985).

實驗流程：

將pFRT/lacZeo質粒轉染細胞，構建Flp-In母細胞株；
將目的基因克隆進pcDNA5/FRT表達載體；
在Flp-In母細胞中共轉pcDNA5/FRT和pOG44質粒，構建出Flp-In表達細胞株；
檢測目的基因的表達。

flp-in系統流程圖

Flp-In?母細胞株構建：
BioVector NTCC Inc.從Invitrogen公司購得Flp-In?-293、Flp-In?-CV-1、Flp-In?-CHO、Flp-In?-BHK、Flp-In?-3T3、Flp-In?-Jurkat，節約客戶的時間，同時也提供其他細胞上的的Flp-In母細胞株構建服務。

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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