Gene Transfer Assessment
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ProteinSimple platforms let you characterize gene editing and transfer efficiency and use significantly lower amounts of proteins or cells than other methods.
Traditional Western blotting is often used to measure Cas9 transfection efficiency and expression uniformity in cell and gene therapy applications, but it often requires millions of cells just to generate the amount of protein needed for analysis. Single-Cell Westerns with Milo can do this with just a few thousand cells.
See how researchers used Single-Cell Western to better understand erythropoiesis by using a CRISPR-Cas9 screen to search for genes impacting the expression of the erythroid marker CD235a/GYPa. Among the validating hits were genes coding for the N6-methyladenosine (m6A) mRNA methyltransferase (MTase) complex. Single-Cell Westerns were then used to track how the m6A-mRNA regulation may affect translation of PAPCP1 and PABPC4.
- N 6-methyladenosine mRNA marking promotes selective translation of regulons required for human erythropoiesis
Daniel A Kuppers, Sonali Arora, Yiting Lim, Andrea R Lim, Lucas M Carter, Philip D Corrin, Christopher L Plaisier, Ryan Basom, Jeffrey J Delrow, Shiyan Wang, Housheng Hansen He, Beverly Torok-Storb, Andrew C Hsieh, Patrick J Paddison
Nat Commun., Oct 2019; 10(1):4596. doi: 10.1038/s41467-019-12518-6.
Monitor purity, heterogeneity, and stability of Cas9 and its variants
Maurice combines the two core techniques used for Cas9 analysis in one simple to use, automated platform. In this poster, learn how Maurice’s imaged capillary isoelectric focusing (icIEF) detection improves detection of Cas9 at low concentrations and generates high-quality protein stability data in less than 10 minutes per sample, and how his capillary electrophoresis sodium dodecyl sulfate (CE-SDS) assay lets you monitor Cas9 purity and heterogeneity.
mRNA Modulation
Flexible, high-performance assays for measuring hemoglobin expression
Although flow cytometry is used to measure expression of various forms of hemoglobin in single red blood cells to evaluate the efficacy of a gene therapy, flow-specific antibodies against hemoglobin targets either don’t exist or result in poor assay performance. In our Application Note, Multiplexed Single-Cell Western Analysis of Red Blood Cells for Biomarker Detection in Blood and Neurodegenerative Disorders, see how Single-Cell Westerns can differentiate between hemoglobin subunits and measure their expression in single red blood cells. The assay is also much more flexible, letting you use commercial, off the shelf reagents that work best for your cell and gene therapy experiments.
Quantitative validation of protein levels
To demonstrate functional proof-of-concept for a gene therapy, Wes provides quantification and validation on a protein level. As an example, researchers at BioMarin and Charles River developed an alternative huntingtin (HTT) mRNA-lowering strategy using therapeutic RNA modulation throughout the Huntington’s disease was tested in the mouse brain. Read the publication to learn more.
- The expanded CAG repeat in the huntingtin gene as target for therapeutic RNA modulation throughout the HD mouse brain
NA Datson, A González-Barriga, E Kourkouta, R Weij, J van de Giessen, S Mulders, O Kontkanen, T Heikkinen, K Lehtimäki, J C. T. van Deutekom
PLoS One, Feb 2017; 12(2):e0171127. doi: 10.1371/journal.pone.0171127.
Solutions for In Vivo Expression Analysis
Get visualization and quantification of transgene expression with RNAscope™ and BaseScope™ assays from ACD. Explore how these assays combine quantitative, molecular measurement, like PCR, with single-cell resolution in the context of intact tissue morphology, and view ACD’s solutions for Gene Therapy/AAV here at acdbio.com/science/applications/research-areas/gene-therapyaav.
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