Harnessing the power of knockout validation for research reproducibility
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Join Dr Hanna Dreja, Abcam’s Principal Quality Control Scientist, as she discusses how knockout cell lines are being used to increase validation standards and support the reproducibility of research across the life science industry.
Webinar objectives:
- Discover the benefits of working with knockout cell lines both as a technical validation tool and to unlock key biological questions
- Gain expert insight on how knockout cell lines are being applied from basic research and target discovery to pharmacogenomics
- See how Abcam are overcoming the common challenges of working with knockout cell lines
Some additional questions asked during the webinar are answered by our CRISPR experts below.
- Are flow cytometry and RNA detection sufficient to confirm CRISPR knockout of a cell surface protein?
CRISPR KO is performed by the insertion or deletion of nucleotides in the target gene sequence. This results in a frameshift in the gene sequence that prevents expression of the complete protein as the translational machinery will hit a premature stop codon. As a result, you end up with a tiny protein that will be degraded.
The resulting mRNA undergoes nonsense-mediated decay, as the cell detects that it is not fully translated. However, this process is not 100% efficient 1.
If you have confidence in your Abs, I would recommend using protein detection to determine if your protein is truly knocked out.
- How do antibodies work in confirming a CRISPR knockout?
To demonstrate that an antibody binds specifically to the protein of interest, one needs to use a negative control – a control sample that does NOT contain this protein. If you have a cell line that normally expresses the protein, and you genetically modify this to no longer express the protein, you have two perfectly matched samples: a positive and a negative control sample. If your antibody is specific, you will no longer detect the signal in the knockout sample.
About the presenter:
Hanna Dreja received a PhD from Imperial College (London) and completed postdoctoral research at several academic institutes (Institut de Génétique Moléculaire de Montpellier (France), Queen Mary’s University (London) and the University of Cambridge). She has developed a broad scientific skill base with a strong focus on antibodies and infectious agents.
As a team leader of the Scientific Quality Control team at Abcam, her mission is to provide products that exceed our customers' high expectations - helping them to discover more, faster. The knockout initiative has been a cornerstone of the recent team successes and was recognized in the prestigious CiteAb award in 2016 and 2020.