Generation of large-scale chromosomal deletions in primary human keratinocytes by Ramin Farhad

Ramin Farhad

UC San Francisco

Large-scale chromosomal deletions occur frequently in the progression of head and neck squamous cell carcinoma. Here, we demonstrate successful introduction of an in vitro model in primary human keratinocytes to study this phenomenon.

ABSTRACT

Large-scale chromosomal deletions frequently occur in the progression of head and neck squamous cell carcinoma (HNSCC). However, the precise contributions of such genetic insults to tumorigenesis have yet to be elucidated. Here, we introduce a methodology for the generation of chromosomal deletions in primary human keratinocytes to establish their roles in HNSCC progression. Large-scale deletions (~1 Mb) were created by transfecting the cells with various CRISPR/Cas9 vectors coding for guide RNAs (gRNAs) that flank two of the commonly deleted regions in HNSCC tumors at the LRP1B and CSMD1 loci. Creation of deletions was confirmed via endpoint PCR and Sanger sequencing, and efficiency of this process was assessed via TaqMan copy number assay qPCR which indicated a 10-45% reduction in the copy number of loci of interest. The edited keratinocytes could be repeatedly passaged without any signs of differentiation. These deletions did not provide the cells with a proliferative advantage in two-dimensional culture when mixed with non-edited cells. Lastly, the cells carrying the deletion show evidence of invasion in organotypic culture assays.

SUBMIT COMMENT OR QUESTION

14 + 15 =