Isolation and Analysis of a Genome-Edited Single-Hepatocyte from a Cas9 Transgenic Mouse Line.

Isolation and Analysis of a Genome-Edited Single-Hepatocyte from a Cas9 Transgenic Mouse Line. Methods Mol Biol. 2019;1874:257-271 Authors: Sakurai T, Kamiyoshi A, Ohtsuka M, Gurumurthy CB, Sato M, Shindo T Abstract The primary cells isolated from the freshly dissected organ are thought to be different from those cultured for a long time in vitro. For instance, hepatocytes isolated in situ from the liver, display the ability to produce albumin, cultured for about a week often tend to cease production of albumin, including loss of proliferation capability. Thus, it is difficult to perform genome editing (i.e., production of genome-edited hepatocytes by in vitro gene delivery) in such cultured cells. Furthermore, hepatic cell lines available so far do not produce albumin and they would also have lost several characteristics of native liver cells. This poses a serious disadvantage when researchers want to study gene expression profiles under specific experimental settings, for example before and after genome editing. However, this demerit can be overcome if genome-editing is performed in situ in liver and single hepatocytes (both genome-edited and wild-type) can be isolated for analysis immediately following transient gene editing. Previously, we demonstrated successful isolation of genome-edited single hepatocytes, using mice expressing systemic Cas9 transgene (called "sCAT" mouse) and by tail-vein-mediated hydrodynamics-based gene del...
Source: Mol Biol Cell - Category: Molecular Biology Authors: Tags: Methods Mol Biol Source Type: research