TITLE:
Generation of pig primary fibroblast cells harboring defective MC4R genes by N-ethyl-N-nitrosourea mutagenesis: A gene-driven, nontransgenic approach to pig improvement
AUTHORS:
Michiharu Sakurai, Shun-ichi Suzuki, Tadashi Furusawa, Satoshi Mikawa, Tomoyuki Tokunaga, Akira Onishi, Takashi Awata
KEYWORDS:
Pig; Fibroblasts; N-Ethyl-N-Nitorosourea; Mutagenesis
JOURNAL NAME:
American Journal of Molecular Biology,
Vol.3 No.3,
July
17,
2013
ABSTRACT: Transgenic pigs have been produced with the aim of
further improving pigs in terms of economic and environmental traits, but
these animals have not been allowed to enter the food chain. As an alternative
approach to generating pigs with novel traits of economic importance that
cannot be introduced by conventional breeding, we propose a strategy for combining in vitro mutagenesis of pig
primary cells with N-ethyl-N-nitrosourea (ENU) and somatic-cell nuclear transfer (SCNT) technology. To explore the feasibility of this
strategy, we treated pig primary fibroblast cells with ENU, estimated the
per-base mutation frequency induced by the mutagen, clonally cultured about
4000 of the mutagenized cells, and screened them for mutation within the coding
region of the melanocortin-4 receptor (MC4R) gene, a key gene in energy
homeostasis. Through this screening, we obtained 14 cell clones, each
harboring a heterozygous base change within the coding region for MC4R.
Of the mutant cell clones, each of two contained a mutant allele encoding MC4R with greatly reduced receptor activity. By SCNT using these cell clones as
donors, pigs harboring mutated MC4R alleles with reduced receptor activity
can be produced. Our strategy for generating pigs with novel genetic traits
likely will be more acceptable to the public than is the use of transgenic
technology.