Comparison of male chimeric mice generated from microinjection of JM8.N4 embryonic stem cells into C57BL/6J and C57BL/6NTac blastocysts.

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. Chimeras were produced by injection of the same JM8.N4 (C57BL/6NTac) derived ES cell line into blastocysts of mixed sex from either C57BL/6J (B6J) or C57BL/6NTac (B6NTac) mice. Similar efficiency of production and sex-conversion of chimeric animals was observed with each strain of blastocyst. However, B6J chimeric males had fewer developmental abnormalities involving urogenital and reproductive tissues (1/12, 8%) compared with B6NTac chimeric males (7/9, 78%). The low sample size did not permit determination of statistical significance for many parameters. However, in each category analyzed the B6J-derived chimeric males performed as well, or better, than their B6NTac counterparts. Twelve of 14 (86%) B6J male chimeras were fertile compared with 6 of 11 (55%) B6NTac male chimeras. Ten of 12 (83%) B6J chimeric males sired more than 1 litter compared with only 3 of 6 (50%) B6NTac chimeras. B6J male chimeras produced more litters per productive mating (3.42 ± 1.73, n = 12) compared to B6NTac chimeras (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.

A survey to establish performance standards for the production of transgenic mice.

The generation of transgenic mice by microinjection of DNA into the pronuclei of fertilized oocytes was described in the early 1980s. A number of parameters affecting the efficiency of the technique were soon identified, including the type of DNA construct, the concentration of DNA being injected, and, most importantly, the strain of mice used for oocyte donors. Since then, hundreds of laboratories and transgenic core facilities across the world have successfully used this technique, essentially as originally described, to create thousands of new transgenic mouse lines. However, the overall procedure continues to be relatively inefficient, in terms of the number of fertilized oocytes required to produce a transgenic mouse, and variations in yields from day to day and construct to construct can be large. Consequently, core facilities often struggle to explain to their customers why a sufficient number of transgenic founders were not produced from a given construct. We believe the field (and individual facilities) would benefit from a rigorous assessment of average yields and expected variations in yields. To this end, we have initiated a survey from the International Society for Transgenic Technologies (ISTT) web site ( www.transtechsociety.org ), to obtain raw microinjection data from as many facilities as possible. We intend to use this data to establish performance standards for the field. Existing facilities will be able to refer to these standards in dealing with dissatisfied clients, and new facilities will be able to aim for an achievable goal. We may even be able to discover an optimum combination of factors that will allow every facility to achieve higher yields.

Tyrosinase Depletion Prevents the Maturation of Melanosomes in the Mouse Hair Follicle.

The mechanisms that lead to variation in human skin and hair color are not fully understood. To better understand the molecular control of skin and hair color variation, we modulated the expression of Tyrosinase (Tyr), which controls the rate-limiting step of melanogenesis, by expressing a single-copy, tetracycline-inducible shRNA against Tyr in mice. Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair. Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis. These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.

Email lists in laboratory animal science.

Email lists can be invaluable for acquiring information that may not be easily accessible in the published literature. The author discusses the general format and functioning of email lists and describes six lists that can be valuable tools for education, training, and information exchange in the field of laboratory animal science.

Lack of maternal glutamate cysteine ligase modifier subunit (Gclm) decreases oocyte glutathione concentrations and disrupts preimplantation development in mice.

Glutathione (GSH) is the most abundant intracellular thiol and an important regulator of cellular redox status. Mice that lack the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH synthesis. Nicotinamide nucleotide transhydrogenase, an inner mitochondrial membrane protein, catalyzes the interconversion of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate; reduced nicotinamide adenine dinucleotide phosphate is required for reduction of GSH disulfide. Previous work supports roles for GSH in preimplantation development. We hypothesized that Gclm-/- mice have increased preimplantation embryonic mortality and that this effect is enhanced by absence of a functioning Nnt gene. Gclm-/- females produced significantly fewer pups per litter than Gclm+/+ littermates. Numbers of oocytes ovulated in a natural estrous cycle or upon superovulation did not differ by genotype. Fewer uterine implantation sites were observed in the Gclm-/- females. Prepubertal Gclm-/- and Gclm+/+ females were superovulated, then mated overnight with a Gclm+/+ male. At 0.5 d postcoitum, Gclm-/- females had significantly lower percentages of zygotes with two pronuclei and higher percentages of zygotes with one pronucleus than Gclm+/+ or Gclm+/- females. At 3.5 d postcoitum, a significantly lower percentage of blastocyst stage embryos was recovered from uteri of Gclm-/- females than Gclm+/+ females. Embryonic development to the blastocyst stage, but not the two-cell stage, was significantly decreased after in vitro fertilization of oocytes from Gclm-/- females compared with Gclm+/+ females. The Nnt mutation did not enhance the effects of Gclm genotype on female fertility. These results demonstrate critical roles for maternal GSH in supporting normal preimplantation development.

Lower skeletal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin.

Mutations in the myostatin gene are associated with hypermuscularity, suggesting that myostatin inhibits skeletal muscle growth. We postulated that increased tissue-specific expression of myostatin protein in skeletal muscle would induce muscle loss. To investigate this hypothesis, we generated transgenic mice that overexpress myostatin protein selectively in the skeletal muscle, with or without ancillary expression in the heart, utilizing cDNA constructs in which a wild-type (MCK/Mst) or mutated muscle creatine kinase (MCK-3E/Mst) promoter was placed upstream of mouse myostatin cDNA. Transgenic mice harboring these MCK promoters linked to enhanced green fluorescent protein (EGFP) expressed the reporter protein only in skeletal and cardiac muscles (MCK) or in skeletal muscle alone (MCK-3E). Seven-week-old animals were genotyped by PCR of tail DNA or by Southern blot analysis of liver DNA. Myostatin mRNA and protein, measured by RT-PCR and Western blot, respectively, were significantly higher in gastrocnemius, quadriceps, and tibialis anterior of MCK/Mst-transgenic mice compared with wild-type mice. Male MCK/Mst-transgenic mice had 18-24% lower hind- and forelimb muscle weight and 18% reduction in quadriceps and gastrocnemius fiber cross-sectional area and myonuclear number (immunohistochemistry) than wild-type male mice. Male transgenic mice with mutated MCK-3E promoter showed similar effects on muscle mass. However, female transgenic mice with either type of MCK promoter did not differ from wild-type controls in either body weight or skeletal muscle mass. In conclusion, increased expression of myostatin in skeletal muscle is associated with lower muscle mass and decreased fiber size and myonuclear number, decreased cardiac muscle mass, and increased fat mass in male mice, consistent with its role as an inhibitor of skeletal muscle mass. The mechanism of gender specificity remains to be clarified.