The wild type ALKBH5-CDS and mutant ALKBH5-CDS were PCR-amplified from pF RT/TO/HIS/FLAG/HA-ALKBH5 plasmid (38073, addgene) and pFLAG CMV5.1-ABH 5-H204A (kindly provided by Dr. Chuan He), and then cloned into the pCDH lentivir al vector (CD513B-1, SBI, Mountain View, CA) using XbaI and BamHI enzyme sites. The TRC shRNAs targeting human ALKBH5 (shA5-#1: TRCN0000291838; shA5-#2: TRCN0000291769), mouse Alkbh5 (shA5-#a: TRCN0000201776; shA5-#b: TRCN00001 92524) were purchased from Sigma-Aldrich, the non-targeting control (pLKO.1) was fr om addgene. The inducible shRNA plasmids (TRIPZ-shA5-#3: V2THS_173653; TRIPZ-shA5-#4: V2THS_173654), as well as the non-targeting control shRNA, were all purchased from GE Dharmacon. The Lenti-iCas9-neo (doxycycline-inducible Cas9-EGFP ve ctor) and lenti-guide (gRNA expression vector) were purchased from Addgene. Lenti-s gALKBH5 was constructed as previously described (Ran et al., 2013 (link)). Stbl3™ E.coli (C7373–03, Thermo Fisher Scientific) and 5-alpha Competent E. coli (C29871, New E ngland Biolabs) were used in transformation.
Lenti icas9 neo
Manufactured by Addgene
The Lenti-iCas9-neo is a lentiviral vector that expresses the Inducible Cas9 (iCas9) protein and a neomycin resistance gene. The iCas9 protein is under the control of a tetracycline-inducible promoter, allowing for temporal regulation of Cas9 expression in target cells.
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Lab products found in correlation
Lentiguide puro, by Addgene (2 mentions)
Lentiguide, by Addgene (1 mentions)
5 alpha competent e coli, by New England Biolabs (1 mentions)
Stbl3 e coli, by Thermo Fisher Scientific (1 mentions)
Plko 1, by Addgene (1 mentions)
Lenticrispr v2, by Addgene (1 mentions)
Ez tet plko puro, by Addgene (1 mentions)
Lenticrispr v2 vector, by Addgene (1 mentions)
Lenti dcas vp64 blast, by Addgene (1 mentions)
Cropseq guide puro, by Addgene (1 mentions)
Lentimph v2, by Addgene (1 mentions)
6 protocols using lenti icas9 neo
1
Recombinant ALKBH5 Plasmid Generation
2
CRISPR-Cas9 Targeting of HBV Genome
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We selected a guide RNA (gRNA) targeting the HBV genome (gRNA21) with reference to a previous report[15 ] and designed control gRNAs targeting neither HBV nor the human genome. The gRNAs were cloned into a lentiviral gRNA/Cas9 expression vector (lentiCRISPR v2; addgene#52961) or a lentiviral gRNA expression vector (lentiGuide‐Puro; addgene#52963). HepG2‐hNTCP‐C4‐iCas9 cells were established through the transduction of a lentiviral vector expressing DOX‐inducible Cas9 (Lenti‐iCas9‐neo; Addgene #85400) into HepG2‐hNTCP‐C4 cells,[23 ] followed by green fluorescent protein selection with fluorescence‐assisted cell sorting.
3
CRISPR Knockouts and Overexpression Constructs
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We cloned individual guide sgRNA vectors for screen validation using the lentiGuide-Puro (Addgene 52963; a gift from Feng Zhang) vector in conjunction with Lenti-iCas9-neo (Addgene 85400; a gift from Qin Yan). For knockouts of human CHUK, MIOS, IRAK1, COPS4, and other validations of the MSK-LX227C suppressor screen, we used the lentiCRISPR v2 vector (Addgene 52961; a gift from the Zhang laboratory). To induce knockdown of GFP, NEDD8, RBX1, INSM1, and COPS4, we used the EZ-Tet-pLKO series of plasmids (EZ-Tet-pLKO-Puro [Addgene 85966; a gift from Cindy Miranti], EZ-Tet-pLKO-Blast [Addgene 85973; a gift from Cindy Miranti], and EZ-Tet-pLKO-Hygro [Addgene 85972; a gift from Cindy Miranti]). shRNA and sgRNA sequences are listed in Supplemental Table S2 . The INSM1 cDNA was a gift from the Peter Nelson laboratory (Origene RG215007). The ASCL1 lentiviral overexpression plasmid (phASCL1-N106; Addgene 31781; a gift from Gerald Crabtree) was used as the base vector for the constitutive neuroendocrine overexpression construct. The INSM1 cDNA was cloned into the phASCL1 vector backbone. To create the empty control (phEmpty), the ASCL1 cDNA was removed and the vector religated.
4
Inducible Klotho Gene Expression System
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Example 7
In the context of gene therapy, inducible gene expression systems may enable gene expression to be triggered at specific times and in specific cell types. To develop a Tet-on system for inducing Klotho gene expression, the ZFP3_VPR construct was cloned into a doxycycline-inducible vector. The ZFP3_VPR and ZFP52_VPR sequences and the inducible vector (Lenti-iCas9-neo, Addgene #85400) were amplified using Clontech HiFi according to the manufacturers protocol and the following primers:
5
Lentiviral Plasmid Construction and Characterization
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The Lenti-idCas9-KRAB-neo and Lenti-idCas9-VP64-neo plasmids were generated from Lenti-iCas9-neo (Addgene, plasmid # 85,400), TRE-KRAB-dCas9-IRES-GFP (Addgene, plasmid # 85,556) and lenti dCAS-VP64_Blast (Addgene, plasmid # 61,425). The lentiMPH v2-EGFP plasmid was generated by replacing hygromycin with EGFP from lentiMPH v2 (Addgene, plasmid # 89,308). CROPseq-i-sgRNA-BFP and CROPseq-a-sgRNA-BFP backbones were generated from CROPseq-Guide-Puro (Addgene, plasmid # 86,708). CROPseq-i-NC-BFP and CROPseq-i-KRAS-BFP plasmids were generated from CROPseq-i-sgRNA-BFP backbone with i-NC or i-KRAS, respectively. CROPseq-a-NC-BFP. CROPseq-a-KRAS-1-BFP and CROPseq-a-KRAS-2-BFP plasmids were generated from CROPseq-a-sgRNA-BFP backbone with a-NC, a-KRAS-1, or a-KRAS-2, respectively. The sgRNA sequences are listed in Supplementary Table 8 . Lenti-iCas9-neo was a gift from Qin Yan, TRE-KRAB-dCas9-IRES-GFP was a gift from Eric Lander, lenti dCAS-VP64_Blast and lentiMPH v2 were gifts from Feng Zhang, and CROPseq-Guide-Puro was a gift from Christoph Bock [35 (link)–39 (link)].
6
Generation of HuH7iCAS9 Cell Line
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HuH7iCAS9 were generated by infection with a lentivirus using standard procedures. Briefly, 293FT cells were transfected with Lenti-iCas9-neo [16] (Addgene plasmid 85400) together with psPAX2 and pMD2.G. Infected HuH7iCAS9 Cells were selected with G418 (0.4 mg/ ml) for 5 days, followed by induction with Doxycycline (5 μg/ml) and cell sorting (FACS Aria III) using the GFP tag. Sorted cells were maintained in low glucose DMEM supplemented with amphoterin/penicillin/streptomycin. Gene editing was initiated by the addition of doxycycline to induce clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 prior to the transfection of plasmids. Single clones were obtained by seeding the cells in 96 well plates. Cells were allowed to proliferate and editing was assessed by Resolvase assay (Guide-It, TAKARA), as recommended by the supplier.
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