221 GENERATION OF GERM-LINE COMPETENT EMBRYONIC STEM CELLS FROM NON-OBESE DIABETIC (NOD) MICE USING A SINGLE INHIBITOR

Abstract

The use of inbred mouse strains, where selective mating and tissue availability can be used to study the role of specific genes during disease pathogenesis is a synergistic approach to human type 1 diabetes (T1D) studies, which are hindered by genetic heterogeneity and inability to obtain relevant tissue biopsies. An excellent model for T1D is the non-obese diabetic (NOD) mouse strain, which spontaneously develops autoimmunity with lymphocytic infiltration of pancreatic islets. Autoimmune disease in NOD mice is multigenic and more than 20 susceptibility loci have been identified. Thus, genetic manipulation of NOD mice via gene targeting in embryonic stem cells (ESC) is a key strategy for identifying the underlying genes for these loci and characterising their roles in disease pathogenesis. Derivation of ESCs from NOD mice has proved to be extremely challenging and the NOD mouse is classified as one of the non-permissive mouse strains for derivation of ESC (Nagafuchi et al. 1999 FEBS Lett. 455, 101–141; Nichols et al. 2009 Nat. Med. 15, 814–818). Using the conventional mouse ESC culture medium - Knockout DMED medium supplemented with 20% Knockout serum replacement (KSR), 2 mM glutamine, 0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol (all from Invitrogen) and 2000 U mL^–1 LIF (Millipore), we efficiently derived robust NOD ESC lines by inhibiting either the ERK or the GSK3b signalling pathway. Nineteen NOD ESC lines following supplementation of the ERK signalling inhibitor SC1 (SC1-NOD ESC) and 10 lines with GSK3b signalling inhibitor BIO (designed as BIO-NOD ESC) from 22 and 12 embryos, respectively (86.3 and 83.3%; P = 0.8). Two SC1-NOD and 2 BIO-NOD ESC were selected for characterisation. All 4 lines expressed the pluripotency-specific genes OCT4, NANOG and SSEA1, showed euploid karyotypes and generated teratomas in SCID mice. Two BIO-NOD ESC lines contributed to chimeric mice after injection into C57BL/6 blastocysts and embryo transfer. Chimeras from the both BIO-NOD ESC lines produced progeny of pure NOD genetics after mating with wild-type NOD females, demonstrating germ-line competence. The NOD ESC lines generated provide a unique tool to study genes implicated in T1D.