The coding regions of mouse Aid (NM_009645.2, NCBI) and mouse Apobec1 (NM_001134391, NCBI) were cloned from mouse ES cells by RT-PCR. The PCR products were sequenced and subcloned into pENTR-D-TOPO (Invitrogen) and recombined with pMXs-gw [1] (link) using LR recombinase according to manufacturer’s instructions (Invitrogen). Mouse dominant-negative Apobec1 (H61K/C93S/C96S) [20] (link) was generated by PCR-based site-directed mutagenesis. To generate lentivirus vectors encoding doxycycline-inducible reprogramming factors, TRE, the Gateway cassette (Invitrogen) and rtTA2s-M2 (Clontech) were introduced into a pLKO.1 backbone (#10878, Addgene). Then coding sequences of Oct3/4, Sox2, Klf4 and c-Myc were inserted by the LR reaction to make pLV-TRE-rtTA2s-M2-Oct3/4, -Sox2, -Klf4 and -c-Myc. psPAX2 (#12260) and pMDG.2 (#12259) were obtained from Addgene. The primers used for the construction of plasmids are listed in Table S7 .
Plko 1 backbone
Manufactured by Addgene
The PLKO.1 backbone is a plasmid vector that can be used for lentiviral gene delivery. It contains the essential elements required for lentivirus production, including the lentiviral long terminal repeat (LTR) sequences, a multi-cloning site for inserting the gene of interest, and a selectable marker. The PLKO.1 backbone serves as a backbone for constructing lentiviral expression vectors, allowing researchers to deliver and express their genes of interest in target cells.
Automatically generated - may contain errors
Lab products found in correlation
Pmdg 2, by Addgene (1 mentions)
Lr recombinase, by Thermo Fisher Scientific (1 mentions)
Gateway cassette, by Thermo Fisher Scientific (1 mentions)
Pmxs gw, by Thermo Fisher Scientific (1 mentions)
Rtta2s m2, by Takara Bio (1 mentions)
Pentr d topo, by Thermo Fisher Scientific (1 mentions)
Pspax2, by Addgene (1 mentions)
Tet on plasmid, by Addgene (1 mentions)
3 protocols using plko 1 backbone
1
Cloning and Reprogramming Factors
2
Generating Inducible Notch1 Constructs
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shRNA plasmids were constructed by inserting annealed targeting oligos into the pLKO.1 backbone (Addgene, 8453) after digestion with AgeI and EcoRI (NEB, R0552 and R0101). ICN1 cDNA was cloned from the Puro-iNotch1IC plasmid (Addgene, 75338)66 (link) for mice and EF.hICN1.CMV.GFP plasmid (Addgene, 17623)67 (link) for humans and then inserted into the tet-on plasmid (Addgene, 80921)68 . An HA tag was also added to the N terminus of ICN1. Routine PCR and Sanger sequencing were performed to confirm successful insertion into the vector. The oligonucleotide sequences used for shRNA knockdown are listed in Supplementary Data 10 .
3
Lentiviral Knockdown of Human EYA2
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Lentiviral clones expressing two or three nonoverlapping shRNAs directed against human EYA2 (TRCN0000051947 and TRCN0000051946) or a nontargeting control shRNA was obtained from Sigma-Aldrich. shRNAs targeting 3′ untranslated region (UTR) of human EYA2 were designed with hairpin design tool in GPP Web Portal (https://portals.broadinstitute.org/gpp/public/seq/search ; Board Institute). Annealed oligos were then inserted into PLKO.1 backbone (Cat# 10878; Addgene) as instructed. 293FT cells were transfected with the transfer plasmids together with the packaging vectors pCMV-dR8.2 dvpr and pCI-VSVG using a standard calcium phosphate transfection method in DMEM plus 10% FBS. 12 h later after transfection, culture medium was changed to complete Neurobasal medium. Medium containing virus particles was collected and centrifuged at 3,000 rpm for 10 min at 4°C. The supernatant was filtered with a 0.45-µm filter for immediate use or stock in −80°C.
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