Cloning:Article Title: Optimization of HITI-Mediated Gene Insertion for Rhodopsin and Peripherin-2 in Mouse Rod Photoreceptors: Targeting Dominant Retinitis Pigmentosa
Article Snippet: Description in the text closed DNA casettes (5' to 3' direction) cloning method Plasmid backbone pCAG-SpCas9 SpCas9 InFusion HD Cloning Plus system (TaKaRa)*1 pCAG (Addgene 11159) pCAG-EGxxFP [EGFP (1-600bp) with stop codon]-[mRho gRNA target]-[EGFP (120-720bp)] InFusion HD Cloning Plus system (TaKaRa)*1 pCAG (Addgene 11159) pCAG-mCherry mCherry InFusion HD Cloning Plus system (TaKaRa)*1 pCAG (Addgene 11159) pCAG-NLS-mCherry NLS, mCherry InFusion HD Cloning Plus system (TaKaRa)*1 pCAG (Addgene 11159) pbRho2k-SpCas9 bRho2k, SpCas9, synthetic pA InFusion HD Cloning Plus system (TaKaRa)*1 pCAG (Addgene 11159) pbRho300-SpCas9 bRho300bp, SpCas9, synthetic pA InFusion HD Cloning Plus system (TaKaRa)*1 pCAG (Addgene 11159) pBAsi-U6-mRho-gRNA mRho-gRNA(1/2/3)*3, gRNA scaffold InFusion HD Cloning Plus system (TaKaRa)*1 pBASi (TaKaRa) pBAsi-U6-mPrph2-gRNA mPrph2-gRNA(1/2/3)*3, gRNA scaffold InFusion HD Cloning Plus system (TaKaRa)*1 pBASi (TaKaRa) (Illustrated in Fig.1C) [mRho-gRNA(1/2/3)]*5-[ChimericIntron, mRhoCDS, FurinP2A, AcGFP, mRho3'UTR]-[mRho-gRNA(1/2/3)]*5 InFusion HD Cloning Plus system (TaKaRa)*1 pLeaklessIII*4 (Illustrated in Fig.1G) [mRho-gRNA1]*5-[ChimericIntron, mRhoCDS, FurinP2A, AcGFP, mRho3'UTR]-[mRho-gRNA1]*5 InFusion HD Cloning Plus system (TaKaRa)*1 pAAV*6 (Illustrated in Fig.2A) [mRho-gRNA1]*5-[ChimericIntron, mRhoCDS, FurinP2A, NLS, AcGFP, mRho3'UTR]-[mRho-gRNA1]*5 InFusion HD Cloning Plus system (TaKaRa)*1 pLeaklessIII*4 (Illustrated in Fig.2E) [mRho-gRNA1]*5-[ChimericIntron, mRhoCDS, FurinP2A, mCherry, mRho3'UTR]-[mRho-gRNA1]*5 InFusion HD Cloning Plus system (TaKaRa)*1 pLeaklessIII*4 (Illustrated in Fig.5A) [mPrph2-gRNA(1/2/3)]*5-[ChimericIntron, mPeph2CDS, FurinP2A, AcGFP, mPrph3'UTR]-[mPrph2-gRNA(1/2/3)]*5 InFusion HD Cloning Plus system (TaKaRa)*1 pLeaklessIII*4 AAV8-SpCas9 bRho300bp, SpCas9, synthetic pA InFusion HD Cloning Plus system (TaKaRa)*1 pAAV*6 AAV8-Donor+gRNA [U6-mRho-gRNA1, gRNA scaffold]-[mRho-gRNA1]*5-[ChimericIntron, mRhoCDS, FurinP2A, AcGFP, mRho3'UTR]-[mRho-gRNA1]*5 InFusion HD Cloning Plus system (TaKaRa)*1 pAAV*6 AAV8-Donor [mRho-gRNA1]*5-[ChimericIntron, mRhoCDS, FurinP2A, AcGFP, mRho3'UTR]-[mRho-gRNA1]*5 InFusion HD Cloning Plus system (TaKaRa)*1 pAAV*6 scAAV8-U6-gRNA×2 [U6-mRho-gRNA1, gRNA scaffold]-[WPRE]-[U6-mRho-gRNA1, gRNA scaffold] InFusion HD Cloning Plus system (TaKaRa)*1 pscAAV (described in Fig.S4) [mRho 5' franking arm]-[loxP]-[stop codon]-[IRES]-[AcGFP]-[b globin polyA]-[loxP]-[mRho 3' franking arm] *7 InFusion HD Cloning Plus system (TaKaRa)*1 pBluescriptII SK- *1 The reagents were used according to the manufacturer’s instructions.
Plasmid Preparation:Article Title: Optimization of HITI-Mediated Gene Insertion for Rhodopsin and Peripherin-2 in Mouse Rod Photoreceptors: Targeting Dominant Retinitis Pigmentosa
Article Snippet: Description in the text closed DNA casettes (5' to 3' direction) cloning method Plasmid backbone pCAG-SpCas9 SpCas9 InFusion HD Cloning Plus system (TaKaRa)*1 pCAG (Addgene 11159) pCAG-EGxxFP [EGFP (1-600bp) with stop codon]-[mRho gRNA target]-[EGFP (120-720bp)] InFusion HD Cloning Plus system (TaKaRa)*1 pCAG (Addgene 11159) pCAG-mCherry mCherry InFusion HD Cloning Plus system (TaKaRa)*1 pCAG (Addgene 11159) pCAG-NLS-mCherry NLS, mCherry InFusion HD Cloning Plus system (TaKaRa)*1 pCAG (Addgene 11159) pbRho2k-SpCas9 bRho2k, SpCas9, synthetic pA InFusion HD Cloning Plus system (TaKaRa)*1 pCAG (Addgene 11159) pbRho300-SpCas9 bRho300bp, SpCas9, synthetic pA InFusion HD Cloning Plus system (TaKaRa)*1 pCAG (Addgene 11159) pBAsi-U6-mRho-gRNA mRho-gRNA(1/2/3)*3, gRNA scaffold InFusion HD Cloning Plus system (TaKaRa)*1 pBASi (TaKaRa) pBAsi-U6-mPrph2-gRNA mPrph2-gRNA(1/2/3)*3, gRNA scaffold InFusion HD Cloning Plus system (TaKaRa)*1 pBASi (TaKaRa) (Illustrated in Fig.1C) [mRho-gRNA(1/2/3)]*5-[ChimericIntron, mRhoCDS, FurinP2A, AcGFP, mRho3'UTR]-[mRho-gRNA(1/2/3)]*5 InFusion HD Cloning Plus system (TaKaRa)*1 pLeaklessIII*4 (Illustrated in Fig.1G) [mRho-gRNA1]*5-[ChimericIntron, mRhoCDS, FurinP2A, AcGFP, mRho3'UTR]-[mRho-gRNA1]*5 InFusion HD Cloning Plus system (TaKaRa)*1 pAAV*6 (Illustrated in Fig.2A) [mRho-gRNA1]*5-[ChimericIntron, mRhoCDS, FurinP2A, NLS, AcGFP, mRho3'UTR]-[mRho-gRNA1]*5 InFusion HD Cloning Plus system (TaKaRa)*1 pLeaklessIII*4 (Illustrated in Fig.2E) [mRho-gRNA1]*5-[ChimericIntron, mRhoCDS, FurinP2A, mCherry, mRho3'UTR]-[mRho-gRNA1]*5 InFusion HD Cloning Plus system (TaKaRa)*1 pLeaklessIII*4 (Illustrated in Fig.5A) [mPrph2-gRNA(1/2/3)]*5-[ChimericIntron, mPeph2CDS, FurinP2A, AcGFP, mPrph3'UTR]-[mPrph2-gRNA(1/2/3)]*5 InFusion HD Cloning Plus system (TaKaRa)*1 pLeaklessIII*4 AAV8-SpCas9 bRho300bp, SpCas9, synthetic pA InFusion HD Cloning Plus system (TaKaRa)*1 pAAV*6 AAV8-Donor+gRNA [U6-mRho-gRNA1, gRNA scaffold]-[mRho-gRNA1]*5-[ChimericIntron, mRhoCDS, FurinP2A, AcGFP, mRho3'UTR]-[mRho-gRNA1]*5 InFusion HD Cloning Plus system (TaKaRa)*1 pAAV*6 AAV8-Donor [mRho-gRNA1]*5-[ChimericIntron, mRhoCDS, FurinP2A, AcGFP, mRho3'UTR]-[mRho-gRNA1]*5 InFusion HD Cloning Plus system (TaKaRa)*1 pAAV*6 scAAV8-U6-gRNA×2 [U6-mRho-gRNA1, gRNA scaffold]-[WPRE]-[U6-mRho-gRNA1, gRNA scaffold] InFusion HD Cloning Plus system (TaKaRa)*1 pscAAV (described in Fig.S4) [mRho 5' franking arm]-[loxP]-[stop codon]-[IRES]-[AcGFP]-[b globin polyA]-[loxP]-[mRho 3' franking arm] *7 InFusion HD Cloning Plus system (TaKaRa)*1 pBluescriptII SK- *1 The reagents were used according to the manufacturer’s instructions.
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