Table 1 Functional RNAi arrayed screenings
From: Arrayed functional genetic screenings in pluripotency reprogramming and differentiation
Targeted genes/RNAi method | Cell model | Screening method | Readouts | No of hits (examples); additional findings. # | Refs. |
|---|---|---|---|---|---|
326 cDNAs of M13 mESC subtracted with MEFs/LT or EP RNAi plasmids | Mouse ESCs | qPCR (DNA Engine Opticon 2 system, Bio-Rad) | qPCR of Oct4 | 8 (Zfp42/Rex-1, Pcna, Tle4, Dppa5, Wdr77, Ash2l, Uble1b). | [60] |
70 mouse genes downregulated upon RA differentiation/LV shRNA | Mouse ESC line CCE | Manual FC | Self-renewal competition assay | 10 (Esrrb, Tbx3, Tcl1, Nanog, Oct4 and Sox2). | [61] |
1008 mouse genes encoding chromatin proteins/LT esiRNAs | Mouse ESC lines | Manual TLM | Defects in viability and alterations in cell or colony morphology | 68, 20 validated (Tip60-p400 complex: Ruvbl1c, Ruvbl2c, Dmap1c, Ep400, Htatipc/Tip60, Trrapc. Yeats4). | [62] |
197 chromatin regulators/LV shRNA (6 per gene) | Mouse ESCs | HCS (ArrayScan HCS Reader, Cellomics). | Average Oct4 staining intensity (Hoechst cells) | 10 (SetDB1/ESET, Ube2i/Ubc9, Ehmt1, Suv39h2, Cbx7, Cbx8/Pc3, Ezh2, Hdac3, Sin3a, Sap18). 3 (Smarcd1, Arid1a, Smarcb1) | [64] |
25,057 mouse genome-scale library/LT esiRNAs | Mouse Oct4-Gip ESCs (Oct4-GFP) | Automated FC (FACSCalibur, BD) | % Oct4-GFP+ cells | 282, 21 validated (Ctr9, Rtf1, Nfya, Ptbp1, Wdr61, Cpsf3, Fip1l1, Iws1, Thoc2, Rnf2, Cxxc1, Cnot1, Ncl, Apc, Ube2m, Shfdg1). | [74] |
16,683 mouse siGenome library (Dharmacon)/LT siRNAs pools | Mouse Oct4-Gip ESCs (Oct4-GFP) | Automated FC (LSRII, BD) | % Oct4-GFP+ cells | 148, 8 validated (Cnot3, Eny2, Fip1l1, Mga, Pcgf6, Pcid2, Smc1a, Trim28). | [75] |
312 mouse genes enriched for gene targets involved in chromatin regulation, early developmental signal transduction, and transcription (Qiagen)/LT shRNA | Mouse Nanog-GFP NG4 ESCs cells | Automated FC (FACS sort or LSR II, BD) | % Nanog-GFP+ cells | 19 (Smad2, Smad4, Ctnnb1, Nfkbia, Nfkbib, Jak1, Gsk3b, Stat3, Esrrb, Oct4, Sall4, Sox2, Rif1). 21 (Fgfr2, Fgfr4, Erk1, Axin2, Hira, Ptma, Raf1, RhoA, Smad7, Socs3, Tcf3, Smarca4, Smarcb1, Smarcc1, Smarce1). | [65] |
& 30,892 cDNAs expression libraries, mouse (7249) and human (3820 from MGC and 19,823 from OriGene Technologies)/LT plasmid | Mouse P19 EC cells (Nanog-luciferase) | Automated L (CLIPR, Molecular Devices) | Nanog-luciferase Activity/Well | 159, 90 validated (Timp2, Hig2, Mki67ip). 70, 14 validated (PU.1/Spi1, Prkaca, Jun). | [84] |
21,121 human whole genome (Dharmacon)/LT siRNAs pools | Human H1 ESCs (Oct4-GFP) | Automated CFM (IXU Ultra, Research Instruments) | % Oct4-GFP+ cells and nuclei (Hoechst) count | 566 (PRMD14, NFRKB, Med12/13/14/17/17/24, CDC42, RRAS, COL4A4, RHOA, CLASP1, AHCTF1, NUP107, TUBB4, PSMD2, ODF2, CENPA, DCTN2, APITD1, CLASP1, CENPQ, RAD9B, RAD17); reduced reprogramming (PRDM14, NFRKB). | [82] |
10,000 mouse genes / LV shRNA | Mouse ESCs | HCS (ArrayScan, Cellomics) | Average Oct4 pixel intensity of cells (nuclei/Hoechst) in the well | 43 (12 Med subunits, Smc1a, Smc3, Stag2, Nipbl). | [76] |
4010 mouse genes, including all signaling genes, transcription factors, and chromatin regulators (Dharmacon)/LT siRNAs pools | $ MEFs Reprograming (Dox-Inducible OKMS cassette) | HCS (InCell Analyzer 1000, GE) | Total iPSC Colony Area (overlap of AP/DAPI staining) | Not specified by authors (Cdh1, Par3 and Crb3, Smad1/4, BMPRII, ALK3). | [87] |
571 kinases and 253 phosphatases/LT siRNA (Ambion Silencer libraries, Applied Biosystems) | Mouse ESCs CGR8 (with a Wnt/β-catenin signaling luciferase) | EnVision 2104 Multilabel Plate Reader (PerkinElmer). | Luciferase intensity | 55 (AKT1, AURKA, CKB, TEC, MTMR6) decreased Wnt/β-catenin signaling. 14 (Dusp9, Dusp23, Dusp28) increased Wnt/β-catenin signaling. | [66] |
319 human genes enriched in human NTera2D1 EC and H9 ESC lines (Dharmacon)/LT siRNA | Human NTera2D1 EC cells | Automated F | Relative cell number (Hoechst Intensity/Well) | 23 (ZIC3, NANOG, SOX2, HMGA1, POU5F1, NR5A2, RBBP9, BIRC5, CDC2). | [81] |
929 mouse kinases and phosphatases/4801 RV shRNAs | Mouse D3 ESCs | Automated SP | Relative cell number (Alamar Blue Intensity/Well), AP/Alamar Blue Ratio | 27 (Nme6 and Nme7). | [67] |
16,873 mouse siGENOME library (Dharmacon) / LT siRNAs pools | @ Mouse Rex1GFPd2 ESCs (rex1-GFP, a Naïve Marker) | Automated FC (LSR II, BD) | Ratio of high (Naïve) to low (Primed) rex1-GFP expressing cells | 130 (esrrb, stat3, ctr9, smc1a). 792, 316 validated (tcf7l1/tcf3, jarid2, dpy30, jun, mbd3, Gli3). | [77] |
640 known and predicted genes from the ubiquitin-proteasome system / LT siRNA pools | Mouse Nanog-GFP NG4 mESCs cells cultured in +LIF media or -LIF + RA media | Automated FC (FACS sort or LSR II, BD) | % Nanog-GFP+ cells | +LIF 20 (Psmd14 and Usp9x, Rbx1, Rfwd2, Rnf12, Ubr5, and Ddb1, Trim28, Phf5a); reduced reprogramming (Psmd14). -LIF + RA 17 Nine (Fbxw7, Rnf152, Rnf31, Rnf8, Socs3, Topors, Rnf36, Tnfrsf25, Ubl5); increased reprogramming (Fbxw7). | [72] |
104 mouse ESC-associated phosphoregulators/LV shRNA | Mouse ESC line CCE | Automated FC (LSR II, BD) | Self-renewal competition assay | 15 (Acvr2a, Aurka, Aurkb, Bub1b, Chek1, Dyrk3, Mapk4, Mapk13, Ppp4c, Ppm1g, Ppp2r1b); reduced reprogramming (Aurka) | |
8296 and 1640 mouse genes, targeting, respectively, druggable genome and transcription factors (QIAGEN) / LT siRNA | @ Mouse Oct4-Gip ESC (Oct4-GFP) in 2i media (Naïve) | Automated F (BioTek Flx800) | Relative number of puromycin-resistant (Oct4+) cells (Alamar Blue Intensity/Well) | 46, 28 validated (Raf1, Apc, Hgs, KRas, Mapksp1, Tcf7l1/Tcf3, Ctbp2, Tsc1/2, Flcn) | [86] |
16,872 mouse siGENOME library (Dharmacon)/LT siRNAs pools | Mouse Nanog-GFP NG4 ESCs cells | HCS confocal (ImageXpress Ultra, Molecular Devices) | % Nanog-GFP+ cells and nuclei (Hoechst) count | 429, 9 validated (Ap2m1, Piwil2, Fbxw2, Helz2, Snai1, Brd4, Chd4, Htatip2, Prmt1); reduced reprogramming (Snail1). 299, 4 Validated (Brd8, Snai2, Hirip3, Ecd); increased reprogramming (Snai2). | [78] |
734 mouse kinase genes/3686 LV shRNA | $ MEFs Oct4-GFP Reprograming (OSKM retroviruses) | Automated CFM (LSM710, Zeiss) | GFP+ iPSC Colony Count | 59 (TESK1, LIMK2, DGKε, PLK2, BMP2K, BMPR2, MAPK1). | [89] |
20 histone demethylases (HDMs)/LV 5 shRNA per gene | Mouse ESCs | Manual TLM | “ESC growth phenotype” and “colony morphology” | 5 (Jmjd2b/Kdm4b, Jmjd2c/Kdm4c); reduced reprogramming. | [63] |
1066 ubiquitin-proteasome system—UPS/LT 4 siRNA for each gene | * Mouse ESCs DTC26 (aMHC-GFP, cardiac-specific promoter of a-myosin heavy chain) | HCS (ImageXpress Micro, Molecular Devices) | Total GFP Area | 10 (Fbx116, Wdr31, Rcbtb2, Stam2, Dpf3, Fbxl20, Cul5, Kcns3, Sumo2, Amfr). | [73] |
652 epigenetic regulators/siRNAs | $ MEFs Reprograming (Dox-induced OSKM) | HCS (InCell Analyzer 1000, GE) | Total iPSC Colony Area (overlap of AP/DAPI staining) | 76 (Trrap, Ccdc101, Taf12, Gcn5; Ezh2, Suz12, Wdr5, Sirt6, Prmt5). | [88] |
12,348 mouse genes/LT esiRNAs | @ Mouse Oct4-GFP EpiSCs line OE7 | Automated FC (FACSCalibur, BD) | % Oct4-GFP+ Naïve mESC | 467, 53 validated (Apc, Ep300, Tox4, Med12/14, Ctr9, Rtf1, Wdr61/82, Cpsf3, Fip1l1, Cnot1/2/3, Tcf3, Cxxc1, Rnf2, Mll2). 149, 54 validated Brd4, Ctnnb1, Ep400, Smc1a, Rad21, Max, Tip60, Trrap). | [80] |
247 RNA-binding proteins—RBPs expressed in mESCs/LT 4 siRNAs for each target; Block-It, Invitrogen) | Mouse R1 ESCs | qRT-PCR (StepOnePlus System, Applied Biosystems) | qPCR for pluripotency (Nanog) and differentiation markers (Fgf5 / embryonic, Cdx2 / extraembryonic) | 16 (Ddx47, Ddx52, Ddx56, Krr1, Nifk, Nol6, Pdcd11, Rbm28,Rrp7a; Cwc15, Hnrnpk, Hnrnpu, Ptbp1, Rbm42, Srsf7/9G8, Tardbp); reduced reprogramming (Krr1, Ddx47). | [70] |
4558 human genes/siRNAs (Ambion / Dharmacon) | Human NANOG-GFP H1 ESCs cultured under 5 differentiation media: -bFGF-TGF-b, TGF-b inhibition, MEK inhibition, PI3K inhibition and Retinoic Acid | HCS confocal (ImageXpress Ultra, Molecular Devices) | % Nanog-GFP+ cells and nuclei (Hoechst) count | From 39 to 182 hits in 5 screening conditions (AURKA, PLK1, CDC25A, CDK1, CCNB1, CCNB2). | [83] |
21,121 human genes targeted by siRNAs (whole-genome Dharmacon SMARTpooled siRNA library)/ | $ Human BJ fibroblast Reprograming (LV OSKM) | HCS confocal (ImageXpress Ultra, Molecular Devices) | TRA-1-60+ area per number of cells. | 557, 39 validated (PPRC1, ENY2, MGA, PWP1, RAD21). 599, 37 validated (SMAD3, ZMYM2, SFRS11, SAE1, ESET). | [93] |
356 putative RNA-binding proteins—RBPs (SMARTpool siGENOME siRNAs)/LT | @ Mouse ESCs with mCherry knocked into mir-290 ∼ 295 locus (Naïve marker) and eGFP into mir-302 ∼ 367 locus (EpiSCs marker) | HCS (INCell Analyzer 2000, GE) | Area of eGFP and mCherry normalized by Hoechst area. | 34 (adar, apex1, eif2a, elavl1, fus) miR-302 reduction / inhibition of the ESC-to-EpiLC transition. 1 (NF45/Ilf2) miR-302 increase. | [71] |