Genomic Analysis of L5178Y tk+/- Cells and their Induced tk-/- Mutant Colonies
Poster Jan 24, 2017
JND Battey, DJ Smart, NS Sierro, D McHugh, P Vanscheeuwijck, MC Peitsch and NV Ivanov
The genetic basis that underpins the mouse lymphoma assay (MLA) is ostensibly well understood; inactivation of the functional thymidine kinase (tk+) allele in L5178Y cells via mutation or deletion induces trifluorothymidine (TFT) resistance and tk-/-mutants can be selected for against a background of tk+/- wild-type cells via TFT-mediated enrichment (Clements. 2000).
• However, despite its widespread use over the past 20 years for hazard identification purposes, few studies have sought to characterise in greater detail the colonies of cells which propagate as a result of mutagenesis in the assay.
• The advent of whole genome sequencing has the potential to enhance mutagenicity testing since genetic changes can now be investigated with high level of detail at the genome level.
• Next generation sequencing (NGS) technology was used to sequence the genomes of:
a) an L5178Y cell line frequently used in MLA studies.
b) Tk-/- mutant colonies induced by two prototypical mutagens in the assay.
• The goal of this pilot study was to exploit NGS technology with a view to shedding new light on the genome of cells used in the MLA, as well as the changes induced as a result of the exposure to mutagens.
When there is a need to quickly analyze samples using a number of different PCR assays, it is likely that optimal conditions for each assay will not be the same. First, different assays often will require different annealing temperatures for their primers. In addition, amplicons may be designed to be of different lengths and therefore require varying durations of the extension step.READ MORE