Table 1.
Functional RNAi arrayed screenings
| 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) | [68, 69] |
| 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] |
Notes: small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), endoribonuclease-prepared siRNAs (esiRNAs), O (Oct4), K (Klf4), M (c-Myc), S (sox2), lentiviral transduction (LV), retroviral transduction (RV), lipid transfection (LT), electroporation (EP), embryonal carcinoma (EC), embryonic stem cell (ESC), induced pluripotent stem cells (iPSCs), mouse embryonic fibroblasts (MEFs), epiblast stem cells (EpiSCs), flow cytometry (FC), spectrophotometry (SP), fluorometry (F), luminometry (L), transmitted light microscopy (TLM), fluorescence microscopy (FM), confocal fluorescence microscopy (CFM), high-content screening (HCS), alkaline phosphatase (AP). # Knockdown (KD) effect was usually associated with loss of pluripotency features, Naïve-Primed transition, differentiation induction or reduced reprograming (“Pluripotency/Reprograming Effectors” or “Differentiation Inhibitors”); however, some studies also identified genes whose KD promoted pluripotency or reduced differentiation (“Pluripotency/Reprograming Repressors” or “Differentiation Inducers,” italicized in the table). Knockdown effect on reprograming efficiency (when evaluated) is mentioned with the specific gene tested in parenthesis. & Gain of function screen. $ Reprograming. * Differentiation. @ Naïve-Primed Transition