To identify ways to improve the efficiency of generating chimeric mice via microinjection of blastocysts with ES cells, we compared production and performance of ES-cell derived chimeric mice using blastocysts from two closely related and commonly used sub-strains of C57BL/6. (2.17 1.33, n=6). Finally, a greater ratio of germline transmitting chimeric males was obtained using B6J blastocysts (9/14; 64%) compared with chimeras produced using B6NTac blastocysts (4/11; 36%). Use of B6J host blastocysts for microinjection of ES cells may offer improvements over blastocysts from B6NTac and possibly other sub-strains of C57BL/6 mice. locus that encodes nicotinamide nucleotide transhydrogenase (NNT) (Toye et al. 2005). This mutation, which arose spontaneously in the production facility at Jackson Laboratory (Bar Harbor, ME) between 1976 and 1984, involves an in-frame deletion of exons 7C11 and a missense (M35T) mutation in the mitochondrial leader peptide sequence that results in reduced expression of mRNA and no functional protein (Toye et al. 2005; Huang et al. 2006). The mutant allele appears to be restricted to the C57BL/6J strain and sub-strains developed from it EHop-016 IC50 after the mutation arose (Mekada et al. 2009; Huang et al. 2006). Strains with the deletion allele include C57BL/6J, C57BL/6JJcl and C57BL/6JmsSlc. Strains not carrying this mutation include C57BL/6NCrl, C57BL/6JEi, C57BL/6JByJ, C57BL/10J, C57L/J, C58/J, FVB/N, C3H/HeJ, DBA/2J, BALB/cJ, CAST/EiJ, SJL/J, SPRET/EiJ, MOLF/EiJ and AKR/J, NOD and 129Sv/J. Based on correspondence from The Jackson Laboratory, the commercial stock of the albino-B6 strain (B6(Cg)-deletion allele. Whether the mutation in in C57BL/6J mice contributes to the increased testis weight, number of Sertoli cells and sperm output in this strain compared to EHop-016 IC50 C57BL/6JByJ and C57BL/10J is currently unknown. As is expressed in many tissues (Hoek and Rydstrom 1988; Arkblad et Rabbit polyclonal to LRIG2 al. 2001) and loss of NNT is associated with increased cellular oxidative stress (Arkblad et al. 2005), we reasoned that wildtype ES cells might contribute differently to tissues of chimeric embryos following their injection into a wildtype blastocyst and that this might be manifest in chimeric animals with differences in developmental abnormalities and breeding performance. Here, we show improvement of multiple parameters of chimeric male mice generated by microinjection of a EHop-016 IC50 sub-line of C57BL/6NTac (B6NTac)-derived JM8.N4 ES cells (Pettitt et al. 2009) into B6J (wild type) blastocysts. Use of B6J blastocysts as a host for production of chimeric mice with B6NTac-derived ES cells may offer improved efficiency for production of germ line transmitting ES cell-derived chimeric mice. MATERIALS AND METHODS Mice Sources, history and diet of mouse strains at vendors – C57BL/6J (?/?; Cat # 000664, F226 (Jan 2010), maintained on diet 5K52 (PMI); Jackson Laboratory, Sacramento, CA). C57BL/6NTac (+/+; Cat # B6-F, B6-M, Taconic, Oxnard, CA. Maintained on NIH#31M diet (Taconic); Hall to Jax in 1948; Jax at F32 to NIH in 1951; NIH at F151 to Taconic in 1991). After arrival at UCI, mice were housed in individually ventilated cages (Techniplast, Philadelphia, PA) provided with Teklad 2920X irradiated diet (Harlan, Indianapolis, IN) and non-acidified tap water locus or alleles using a three primer, two allele PCR assay that discriminates between the wild-type allele of (B6NTac) and the mutant allele lacking exons 7C11 in B6J mice (Nicholson et al. 2010). The coding sequence for aminoglycoside transferase EHop-016 IC50 (in total DNA extracted from chimeric animals To enable reproducible pipetting of DNA, an aliquot of each genomic DNA was sheared by passing ten times through a 22g needle attached to a 1ml syringe barrel. To analyze the relative contribution of ES cells to different tissues from EHop-016 IC50 each chimera, we quantified the amount of target, derived from the JM8_2H5 ES cell.