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