Application

Functional Genomics/Screening/Target Validation

General description

The Lenti CRISPR Human HPRT1 Positive Control Transduction Particles (LV04 vector) product is a critical positive control to monitor transduction efficiency for lentiCRISPRs. This control is produced from the sequence-verified CRISPR lentiviral plasmid targeting human HPRT1. The protein encoded by this gene is a transferase which plays a central role in the generation of purine nucleotides. Cells with intact HPRT1 will die upon exposure to 6-TG (Product No. A4882), an antimetabolite used in the treatment of leukemias. Conversely, cells with HPRT1 knocked out will increase over time with 6-TG selection. This targets a validated HPRT1 CRISPR site, which serves as an experimental control for lenti CRISPRs. The lenti CRISPR HPRT1 positive control uses a dual-component system consisting of a U6-driven guide RNA plasmid targeting the human HPRT1 gene and hPGK-driven Puromycin and BFP expression. This control requires Cas9 be delivered by a separate vector. Lentiviral-based particles permit efficient infection and integration of the construct into differentiated and non-dividing cells, such as neurons and dendritic cells, overcoming low transfection and integration difficulties when using these cell lines. Self-inactivating replication incompetent viral particles are produced in packaging cells (HEK293T) by co-transfection with compatible packaging and envelope plasmids.

Legal Information

CRISPR Use License AgreementLentiviral, WPRE, and Evrogen Licenses

Other Notes

Ampicillin and puromycin antibiotic resistance genes provide selection in bacterial or mammalian cells, respectively.