...Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with lentiCas9-Blast...A549 cells stably expressing Cas9. A549 cells were transduced with lentiCas9-Blast 1 and then selected with... express Cas9 and are included as a control (WT). Vinculin is a loading control. 1 lentiCas9-Blast was...additional 3 weeks under blasticidin selection. Anti-Cas9 Western blotting was performed on whole-cell extracts...Blog Posts Addgene Protocols Viral Service Introduction This protocol describes how to generate a monoclonal...Selective pressure on this heterogeneous cell pool could lead to reduced transgene expression over time, as...more likely to retain stable transgene expression. Other methods of generating a monoclonal cell line include...

...Deletions with CRISPR Generate genomic deletions in mammalian cells lines using CRISPR/Cas9 Genomic Deletions... Educational Resources Addgene Videos Addgene Videos Browse Addgene's collection of video content...using Addgene's resources, and career advice. For written lab protocol material, visit our Addgene Protocols...Storing and Handling Addgene Plasmids What to do after you receive your plasmids from Addgene Instructions for...samples from Addgene Technology Transfer How to Order A step-by-step walkthrough of Addgene's ordering process...this first installment of the Addgene Careers series, we sit down with Addgene Outreach Scientist Jessica...she loves about working at Addgene! Maria Soriano Maria Soriano, from Addgene's UK office, discusses her...

...Protocol to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector...This protocol can be used to generate stable cell lines expressing a gene of interest from an integrated...A549 cells stably expressing Cas9. A549 cells were transduced with lentiCas9-Blast and then selected with... Protocols Generating Stable Cell Lines with Lentivirus Generating Stable Cell Lines with Lentivirus... may have 1 copy of the transgene, while others have >1 copy of the transgene in their genome. This can...high levels of the transgene may have reduced growth rates, especially if that transgene is toxic. Eventually...low-level transgene expressors may take over the culture. To overcome this, consider generating monoclonal...

...downstream applications. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that...adherent cells) 0.45 μm polyethersulfone filter, Nalgene, 565-0010 (for viral preps, if prep was not previously...transduced with a range of dilutions of pHAGE-TO-dCas9-3XmCherry . 72 h post transduction, cells were assayed...

...Use this protocol to generate lentivirus...downstream applications such as stable-cell line generation. Workflow Timeline Day 0: Seed 293T packaging...adherent cells) 0.45 μm polyethersulfone filter, Nalgene, 565-0010 Microcentrifuge tubes, Neptune 3745.X...per 10 cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA 27.8 μg), this would...

..., BD BD300912 0.45 μm polyethersulfone filter, Nalgene, 565-0010 (for viral preps, if prep was not previously...serial dilutions of the lentiviral vector pHAGE EF1α dCas9-KRAB . The following day, cells were treated with...