ez tet plko hygro vector  (Addgene inc)

Journal: bioRxiv

Article Title: Molecular consequences of acute versus chronic CDK12 loss in prostate carcinoma nominates distinct therapeutic strategies

doi: 10.1101/2024.07.16.603734

Figure Lengend Snippet: (a) Stable CDK12(-) cells show fewer upregulated intronic APAs. Table with the number of APA sites down (DN), no change (NC), or up (UP) in isogenic paired models (CRISPR KO clones vs parental, or 189.4-CDK12 vs 189.4-vec). The ratio UP/DOWN indicates skew towards the IPA phenotype. (b) Validation of Tet-shRNA lines. Western blot with lysates from Tet-shRNA lines treated four days -/+ 100ng/mL doxycycline. (c) LuCaP189.4_CL cells are RAD51 competent. Irradiation and immunostaining (same as in ). Cells were exposed to 6Gy IR and fixed at 3h. Immunofluorescence staining was performed for γH2A.X and RAD51 and images were acquired by confocal microscopy. Left: representative images (white: DAPI, green: γH2A.X, purple: RAD51). Right: quantification of images (∼200-500 cells analyzed per treatment). Line is at mean and significance was determined by unpaired t-test (Mann-Whitney). (d-e) CDK12 knockdown does not prevent RAD51 foci. Additional immunostaining (same as in ) using LNCaP (d) and Skov3 (e) with Tet-shCDK12 or Tet-shBRCA2. Cells were treated four days -/+ dox. Graphs show mean with significance determined by one-way ANOVA (Kruskal-Wallis).

Article Snippet: Tet-inducible shRNA vectors were cloned as previously described( ) into EZ-Tet-pLKO-Hygro (Addgene 85972).

Techniques: CRISPR, Clone Assay, Biomarker Discovery, shRNA, Western Blot, Irradiation, Immunostaining, Immunofluorescence, Staining, Confocal Microscopy, MANN-WHITNEY, Knockdown

Journal: BMC Biotechnology

Article Title: A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector

doi: 10.1186/s12896-017-0341-x

Figure Lengend Snippet: Vector maps and PEG purification. a Basic vector maps (not to scale) for the original Tet-pLKO-Puro vector and our modified versions. b Agarose gel electrophoresis comparing DNA precipitation methods. 10 μg of EZ-Tet-pLKO vector DNA was co-digested with NheI + EcoRI. The digest was split into three 3 μg aliquots and precipitated with isopropanol (Iso) or polyethylene glycol (PEG) at 6 or 8% concentration. 1 μg of control DNA (uncut and cut) was run alongside 1/3 of the precipitated DNA samples

Article Snippet: The EZ-Tet-pLKO-Hygro vector was made by PCR subcloning the Hygro resistance gene from the pGL4.15 vector (Promega) using the following primers: 5′-ATTATGGATCCATGAAGAAGCCCGAACTC and 5′- ATTATGACGTCTTAAACTCGACCTACCTC.

Techniques: Plasmid Preparation, Purification, Modification, Agarose Gel Electrophoresis, Concentration Assay

Journal: BMC Biotechnology

Article Title: A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector

doi: 10.1186/s12896-017-0341-x

Figure Lengend Snippet: Screening techniques. a Diagram showing expected products from PCR screening pLKO ligation-transformed colonies. b Agarose gel (2%) with a positive and negative PCR product. c Vector maps (not to scale) with XhoI and SpeI restriction digest sites labeled in bp. Asterisks indicate corresponding bands in Fig. 3d and e. d Diagram showing expected DNA fragments and relative intensity on gel from an XhoI ( blue ) vs SpeI ( red ) shRNA loop restriction digest screen of the plasmids shown in 3c (i - parental EZ- Tet-pLKO vector with stuffer (Vec + stuff), ii - EZ-Tet-pLKO with shRNA XhoI loop (Vec + sh(X)), iii - EZ-Tet-pLKO with shRNA SpeI loop (Vec + sh(S)). (*) is the predicted 348 bp XhoI fragment spanning the stuffer region in the original Tet-pLKO vector (i). In the EZ-Tet-pLKO vector harboring an shRNA with an XhoI site in the loop (ii), XhoI digestion will generate three small fragments, 190 bp (**), 138 bp (***), and 43 bp (****). In the EZ-Tet-pLKO vector harboring an shRNA with an SpeI site in the loop (iii), SpeI digestion will generate a clearly visible diagnostic 500 bp fragment. e Agarose gel (2%) with XhoI or SpeI shRNA screens of constructs indicated in 3c (i, ii, iii). Each lane was loaded with 4 μg of digested DNA. Bottom image shows lower part of the same gel with a longer exposure to show the barely detectable 43 bp (****) fragment

Article Snippet: The EZ-Tet-pLKO-Hygro vector was made by PCR subcloning the Hygro resistance gene from the pGL4.15 vector (Promega) using the following primers: 5′-ATTATGGATCCATGAAGAAGCCCGAACTC and 5′- ATTATGACGTCTTAAACTCGACCTACCTC.

Techniques: Ligation, Transformation Assay, Agarose Gel Electrophoresis, Plasmid Preparation, Labeling, shRNA, Diagnostic Assay, Construct

Journal: BMC Biotechnology

Article Title: A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector

doi: 10.1186/s12896-017-0341-x

Figure Lengend Snippet: Dox titration and recovery. a Immunoblot showing Dox titration with iPrECs containing EZ-Tet-pLKO-sh.p38α. Cells were treated with Dox for 72 h and lysed. Note: the lower band ( arrow pointing ) is p38α. b Cells were treated −/+ Dox (50 ng/mL) for 72 h. At that time, two samples were lysed (72 h pre-treated) while another plate of treated cells was split and allowed to recover without Dox for 1–8 days. Note: due to changes in confluency, the ‘pre-treated’ cells have higher basal level of p38 (α and δ) than at day 8

Article Snippet: The EZ-Tet-pLKO-Hygro vector was made by PCR subcloning the Hygro resistance gene from the pGL4.15 vector (Promega) using the following primers: 5′-ATTATGGATCCATGAAGAAGCCCGAACTC and 5′- ATTATGACGTCTTAAACTCGACCTACCTC.

Techniques: Titration, Western Blot

Journal: BMC Biotechnology

Article Title: A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector

doi: 10.1186/s12896-017-0341-x

Figure Lengend Snippet: Reagent cost analysis

Article Snippet: The EZ-Tet-pLKO-Hygro vector was made by PCR subcloning the Hygro resistance gene from the pGL4.15 vector (Promega) using the following primers: 5′-ATTATGGATCCATGAAGAAGCCCGAACTC and 5′- ATTATGACGTCTTAAACTCGACCTACCTC.

Techniques: Plasmid Preparation, shRNA, Clone Assay, Ligation

Journal: BMC Biotechnology

Article Title: A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector

doi: 10.1186/s12896-017-0341-x

Figure Lengend Snippet: Cost/benefit comparison of lentiviral shRNA methods

Article Snippet: The EZ-Tet-pLKO-Hygro vector was made by PCR subcloning the Hygro resistance gene from the pGL4.15 vector (Promega) using the following primers: 5′-ATTATGGATCCATGAAGAAGCCCGAACTC and 5′- ATTATGACGTCTTAAACTCGACCTACCTC.

Techniques: shRNA, Clone Assay