pgbd5 plasmids  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Cognate restriction of transposition by piggyBac- like proteins

doi: 10.1093/nar/gkab578

Figure Lengend Snippet: Alignment of piggyBac and PGBD5 protein sequences as described in the Materials and Methods section. Identical amino acids are highlighted in green. The DDD catalytic motif of piggyBac is marked with black bars above the amino acids. The CRD of piggyBac is underlined in red and then expanded to demonstrate the bipartite nuclear localization sequence (underlined black) and cystine residues of the CRD (highlighted red). PGBD5, version 1; PGBD5.2, version 2.

Article Snippet: PGBD5 plasmids were obtained from Addgene including pINDUCER21-PGBD5 (#78121) and pRecLV103-GFP-PGBD5 (#65409) ( 13 ).

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: Cognate restriction of transposition by piggyBac- like proteins

doi: 10.1093/nar/gkab578

Figure Lengend Snippet: The pTpB piggyBac transposon is only excised and integrated by piggyBac in human cells. (A) Schematic of pTpB transposon conferring neomycin resistance. (B) Excision assay using pCMV-SB, -PB, -HyPBase, -pBat or -PGBD5 (pRecLV103-GFP-PGBD5) to express the putative transposase in HEK293 cells transfected with pTpB. The expected excision product is 539 bp. Shown is representative of three independent experiments. (C) Colony count analysis of various transposon:transposase (1 μg:1 μg, 1.8 μg:200 ng or 200 ng:1.8 μg pTpB:transposase) ratios in HEK293 cells using pUC (negative control) or PB/PGBD5; N = 3 ± SD; *, P< 0.05. (D) qPCR of copies of piggyBac ITR/RNAse P of 1 μg:1 μg pTpB:transposase stably transfected HEK293 cells; N = 2 (in triplicate) ± SD; *, P< 0.05 compared to pUC control.

Article Snippet: PGBD5 plasmids were obtained from Addgene including pINDUCER21-PGBD5 (#78121) and pRecLV103-GFP-PGBD5 (#65409) ( 13 ).

Techniques: Excision Assay, Transfection, Negative Control, Stable Transfection, Control

Journal: Nucleic Acids Research

Article Title: Cognate restriction of transposition by piggyBac- like proteins

doi: 10.1093/nar/gkab578

Figure Lengend Snippet: piggyBat transposons are only integrated by piggyBat in human cells. (A) Schematic of pTpBat transposon conferring neomycin resistance. (B) Colony count analysis measuring integration efficiency of pTpBat in HEK293 cells by pUC (negative control), pCMV-PB, pRecLV103-GFP-PGBD5, or pCMV-pBat; N = 3 ± SD; *, P< 0.05.

Article Snippet: PGBD5 plasmids were obtained from Addgene including pINDUCER21-PGBD5 (#78121) and pRecLV103-GFP-PGBD5 (#65409) ( 13 ).

Techniques: Negative Control

Journal: Nucleic Acids Research

Article Title: Cognate restriction of transposition by piggyBac- like proteins

doi: 10.1093/nar/gkab578

Figure Lengend Snippet: piggyBac, but not PGBD5, binds the piggyBac ITR using reporter readout of protein-DNA interaction in transfected cells. (A) Schematic demonstrating that transposase (PB (+ control), GFP (− control) or PGBD5 fused to a VPR activation domain. If binding to the LE (left end) or flipped LE (rLE, reverse LE) ITR occurs, luciferase is expressed and can be measured by IVIS imaging and quantified. (B) IVIS imaging of transfected cells. (C) Photons/s (p/s) readout of transposase ITR interaction; N = 3 ± SD. *, P< 0.05 compared to GFP control.

Article Snippet: PGBD5 plasmids were obtained from Addgene including pINDUCER21-PGBD5 (#78121) and pRecLV103-GFP-PGBD5 (#65409) ( 13 ).

Techniques: Transfection, Control, Activation Assay, Binding Assay, Luciferase, Imaging

Journal: Nucleic Acids Research

Article Title: Cognate restriction of transposition by piggyBac- like proteins

doi: 10.1093/nar/gkab578

Figure Lengend Snippet: Western blot analysis confirms correct expression of piggyBac and PGBD5 in HEK293 cells. HA-PGBD5v1 and v2 have expected molecular weight of 52 and 58 kDa, respectively. The pInducer plasmids lack an HA tag and serve as negative control. HA-tagged (sodium hydrogen exchanger 3) NHE3 and PB serve as positive controls. Blot demonstrates HA (green) with B actin (red) serving as a loading control confirming equal total protein input for each lane.

Article Snippet: PGBD5 plasmids were obtained from Addgene including pINDUCER21-PGBD5 (#78121) and pRecLV103-GFP-PGBD5 (#65409) ( 13 ).

Techniques: Western Blot, Expressing, Molecular Weight, Negative Control, Control

Journal: Nucleic Acids Research

Article Title: Cognate restriction of transposition by piggyBac- like proteins

doi: 10.1093/nar/gkab578

Figure Lengend Snippet: piggyBac, and not PGBD5, integrates PB-EF1-NEO, μpTpB and iμpTpB (i, inverted) transposon vectors. (A) Schematic of μpTpB with shortened ITRs, and iμpTpB with shortened ITRs in flipped orientation (to correspond to PB-EF1-NEO), conferring neomycin resistance. (B) Colony count analysis of G418 resistance in HT-1080 cells transfected with PB-EF1-NEO and pUC, pCMV-SB, pCMV-pB, or pRecLV103-GFP-PGBD5; N = 3 ± SD; *, P< 0.05. (C) Colony count analysis after transfection of the various transposon and transposase vectors into HT-1080 cells and subsequently selected with G418; N = 3 ± SD; *, P< 0.05. (D) qPCR of copies of piggyBac ITR/RNAse P of 1 μg:1 μg pTpB:transposase stably transfected HT-1080 cells; N = 2 (in triplicate) ± SD; *, P< 0.05 compared to pUC control.

Article Snippet: PGBD5 plasmids were obtained from Addgene including pINDUCER21-PGBD5 (#78121) and pRecLV103-GFP-PGBD5 (#65409) ( 13 ).

Techniques: Transfection, Stable Transfection, Control

Journal: Nucleic Acids Research

Article Title: Cognate restriction of transposition by piggyBac- like proteins

doi: 10.1093/nar/gkab578

Figure Lengend Snippet: Plasmid rescue of transposon integration sites in the human genome

Article Snippet: PGBD5 plasmids were obtained from Addgene including pINDUCER21-PGBD5 (#78121) and pRecLV103-GFP-PGBD5 (#65409) ( 13 ).

Techniques: Plasmid Preparation