Generation of Ugt1-Deficient Murine Liver Cell Lines Using TALEN Technology

• DOI: 10.1371/journal.pone.0104816
• Publication Date: 2014-08-13T22:14:52Z
• Authors: Fabiola Porro; Luka Bockor; Alessia De Caneva; Giulia Bortolussi; Andrés F. Muro
• Keywords: Indel

The Crigler-Najjar Syndrome Type I (CNSI) is a rare genetic disorder caused by mutations in the Ugt1a1 gene. It characterized unconjugated hyperbilirubinemia that may result severe neurologic damage and death if untreated. To date, liver transplantation only curative treatment. With aim of generating mutant cell lines Ugt1 gene, we utilized TALEN technology to introduce site-specific exon 4. We report fast efficient method perform gene knockout tissue culture cells, based on use pairs targeting restriction enzyme (RE) sites region interest. This strategy overcame presence allele-specific single nucleotide polymorphisms (SNPs) pseudogenes, conditions limit INDELs' detection Surveyor. obtained liver-derived murine N-Muli clones having INDELs with efficiency close 40%, depending pair RE target site. Sequencing locus WB analysis isolated showed high proportion biallelic cells treated most pair. Ugt glucuronidation activity was reduced basal levels clones. These could be very useful tool study biochemical aspects more natural context, such as substrate specificity, requirement specific co-factors, inhibitors other pharmacological aspects, correlate adding back variants In addition, since genome editing has recently emerged potential therapeutic approach cure diseases, definition an important step towards setting up platform CNSI.