ORF expression in Mammalian cells is very useful for many applications: protein production with HEK293, over-expression of tagged protein for immuno-tracking or immuno-precipitation, development of a cell line for drug discovery such as a cancer model expressing PD-1 or PD-L1…

Unfortunately, plasmid delivery into primary cells and cell lines is often challenging, providing low or even no transfection efficiency. The solution is a robust lentiviral system.

So, how do you identify the right lentiviral system? With the titer! The higher is the better. 10⁷TU/ml is ok… and 10⁸TU/ml is very good.

Are you looking for a simple means to boost the efficiency of gene editing and also to reduce the off-target? Yes, it does exist – in one word: 4-hydroxy Tamoxifen. That’s the HIT-CAS9.

Hybrid drug inducible CRISPR/Cas9 technology, HIT-Cas9

Zhao et al. revealed tight and effective drug control of the CRISPR Cas9 based on 4-Hydroxytamoxifen.

They built a fusion between a mutated human estrogen receptor (ERT2) and the Cas9 endonuclease, which makes the CRISPR Cas9 dependent on 4-hydroxytamoxifen.

Optimal knock-down can be done by a shRNA sequence that depends on the gene expression level and the cell type. Usually, 4 shRNA should be tested to find the one inducing a minimum of 70% increase of the targeted mRNA pool. Genecopoeia has developped shRNA expression vectors with improved performances.

Gemma N. Jones et al has recently published in the British Journal of Cancer an article revealing pRAD50 (Ser635) as a pharmacodynamic biomarker of ATM (AZD0156) and ATR inhibitors (AZD6738) to treat cancers. doi:10.1038/s41416-018-0286-4

Key sensors of DNA damage such as ATM and ATR are targets of choice for drug discovery. A useful screening method is based on synthetic lethality. The pre-screening can be done on convenient HeLa cells.