Contributions
Abstract: EP622
Type: E-Poster Presentation
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
Robust and efficient protocols for gene editing and expression are highly needed in order to facilitate study of gene function in primary tumour cells from patients with chronic lymphocytic leukemia (CLL). CLL cells are notoriously prone to apoptosis when cultured in vitro and are for being hard-to-manipulate. This makes it challenging to modulate their endogenous gene expression or to induce expression of exogenous genes. Conventional methods based on plasmid/siRNA transfection or viral transduction are not efficient in CLL cells.
Aims
Hence, the development of optimized methods adapted to CLL cells gives an opportunity of developing screening platforms to assess function of multiple genes in assays in vitro.
Methods
In this work we set up conditions for CRISPR/Cas9-based gene knockout and mRNA-driven gene overexpression in B cells freshly isolated from peripheral blood of CLL patients. We also developed a platform for high-throughput in vitro production of mRNA encoding multiple genes allowing to scale up eventual screening assays.
Results
We show, on one hand, that it is possible to set up electroporation conditions that only marginally affect CLL cell viability. At such conditions, electroporating CLL cells with ribonucleoprotein complexes containing recombinant Cas9 nuclease and synthetic guides one can achieve >70-80% of homozygous knockout of a given. On the other hand, electroporation with in vitro transcribed mRNA allows to achieve >90% of electroporation efficiency and finely modulate gene expression levels in CLL cells. Gene overexpression persists for up to 4 days and longer allowing to perform long-term in vitro assays.
Conclusion
CRISPR/Cas9- and mRNA-based platforms for gene editing and expression are readily applicable to minimally manipulated primary CLL cells.
Keyword(s): Gene deletion, Gene expression, Screening
Abstract: EP622
Type: E-Poster Presentation
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
Robust and efficient protocols for gene editing and expression are highly needed in order to facilitate study of gene function in primary tumour cells from patients with chronic lymphocytic leukemia (CLL). CLL cells are notoriously prone to apoptosis when cultured in vitro and are for being hard-to-manipulate. This makes it challenging to modulate their endogenous gene expression or to induce expression of exogenous genes. Conventional methods based on plasmid/siRNA transfection or viral transduction are not efficient in CLL cells.
Aims
Hence, the development of optimized methods adapted to CLL cells gives an opportunity of developing screening platforms to assess function of multiple genes in assays in vitro.
Methods
In this work we set up conditions for CRISPR/Cas9-based gene knockout and mRNA-driven gene overexpression in B cells freshly isolated from peripheral blood of CLL patients. We also developed a platform for high-throughput in vitro production of mRNA encoding multiple genes allowing to scale up eventual screening assays.
Results
We show, on one hand, that it is possible to set up electroporation conditions that only marginally affect CLL cell viability. At such conditions, electroporating CLL cells with ribonucleoprotein complexes containing recombinant Cas9 nuclease and synthetic guides one can achieve >70-80% of homozygous knockout of a given. On the other hand, electroporation with in vitro transcribed mRNA allows to achieve >90% of electroporation efficiency and finely modulate gene expression levels in CLL cells. Gene overexpression persists for up to 4 days and longer allowing to perform long-term in vitro assays.
Conclusion
CRISPR/Cas9- and mRNA-based platforms for gene editing and expression are readily applicable to minimally manipulated primary CLL cells.
Keyword(s): Gene deletion, Gene expression, Screening