The International Mouse Phenotyping Consortium (IMPC) is a worldwide effort producing and phenotyping knockout mouse lines to expose the pathophysiological roles of all genes in human diseases and make mice and data available and accessible to the global research community. It has created new knowledge on the function of thousands of genes for which little to anything was known. This new knowledge has informed the genetic basis of rare diseases, posited gene product influences on common diseases, influenced research on targeted therapies, revealed functional pleiotropy, essentiality, and sexual dimorphism, and many more insights into the role of genes in health and disease. Its scientific contributions have been many and widespread, however there remain thousands of "dark" genes yet to be illuminated. Nearing the end of its current funding cycle, IMPC is at a crossroads. The vision forward is clear, the path to proceed less so.

Although thousands of genetically modified mouse strains have been cryopreserved by sperm freezing, the likelihood of cryorecovery success cannot be accurately predicted using conventional sperm parameters. The objective of the present study was to assess the extent to which measurement of a sperm DNA fragmentation index (DFI) can predict sperm quality and fertility after cryopreservation. Using a modified TUNEL assay, we measured and correlated the DFI of frozen-thawed sperm from 83 unique mutant mouse strains with sperm count, motility and morphology. We observed a linear inverse correlation between sperm DFI and sperm morphology and motility. Further, sperm DFI was significantly higher from males with low sperm counts compared to males with normal sperm counts (P < 0.0001). Additionally, we found that viable embryos derived using sperm from males with high DFI (62.7 ± 7.2% for IVF and 73.3 ± 8.1% for ICSI) failed to litter after embryo transfer compared to embryos from males with low DFI (20.4 ± 7.9% for IVF and 28.1 ± 10.7 for ICSI). This study reveals that measurement of DFI provides a simple, informative and reliable measure of sperm quality and can accurately predict male mouse fertility.

The Roux-en-Y Gastric Bypass (RYGB) mouse model is a vital tool for studying the pathophysiology of bariatric surgery and contributes greatly to research on obesity and diabetes. However, complications including postsurgical hypoglycemia can have profoundly negative effects. Unlike in humans, blood glucose (BG) is not typically managed in postoperative rodents, despite their critical role as translational models; without this management, rodents can experience hypoglycemia, potentially impairing wound healing, decreasing survivability, complicating interpretation of research data, and limiting translational utility. In this project, we sought to identify an optimal method for minimally invasive administration of dextrose in C57BL/6N (n = 16; 8 male, 8 female) mice. To do so, we characterized BG pharmacokinetic profiles after subcutaneous and oral-transmucosal (OTM) administration of dextrose. Compared with OTM dosage, the subcutaneous route provided more consistent and reliable delivery of glucose and did not cause significant adverse reactions. We then evaluated the frequency of hypoglycemic events after RYGB in C57BL/6N mice (n = 16; 8 male, 8 female) and the effects of subcutaneous dextrose supplementation on morbidity and mortality. BG measurement and behavioral pain assessment (grimace test) were performed for 3 d after surgery. Hypoglycemic (BG ≤ 60 mg/dL) animals were assigned to dose (5% dextrose SC) or no-dose treatment groups. Nearly all (87%) mice became hypoglycemic; 2 of these mice died. No significant intergroup difference in grimace score or mortality was detected. Overall, our results demonstrate that hypoglycemia is a frequent adverse event after RYGB in mice and that subcutaneous injection of dextrose is a safe and effective way to manage hypoglycemia. Further studies are necessary to optimize the intervention threshold and optimal dosage; regardless, we recommend glycemic management after RYGB surgery in mice.

Different protocols incorporating methyl-β-cyclodextrin (MBCD) and reduced glutathione (GSH) have been reported to improve IVF recovery of cryopreserved mouse sperm on a C57BL/6 (J and N) genetic background. However, it is not clear which IVF protocol is most appropriate when using the various methods to cryorecover sperm with different sperm quality and sample volumes. Therefore, in the present study we correlated sperm motility with fertilization rate and compared the efficiency of different IVF methods using various sperm samples so as to establish general guidelines for mouse sperm cryorecovery by IVF. High linear correlation between sperm fertilization rate and progressive motility was found, R² was 0.9623 and 0.9993 for pre-freezing and post-thaw progressive motility, respectively. High amounts of cryoprotective agent (CPA) were observed to impair both sperm capacitation and fertilization. Moreover, the presence of a large number of immotile sperm in the sperm-oocyte co-incubation drop was found to reduce IVF success which could be partially reversed by supplementation using monothioglycerol (MTG) during centrifugation. It was concluded that the efficiency of IVF using cryorecovered mouse sperm in media containing MBCD and GSH can be predicted from sperm progressive motility. High concentrations of CPA and immotile sperm should be mitigated prior to IVF. The optimum IVF method should be selected based on sperm sample volume and sperm parameters.

CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. Here, we provide an optimized protocol covering sgRNA synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher with basic embryo-manipulation skills can obtain genetically modified mice in 6 weeks. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species.

The breakdown of triglycerides, or lipolysis, is a tightly controlled process that regulates fat mobilization in accord with an animal's energy needs. It is well established that lipolysis is stimulated by hormones that signal energy demand and is suppressed by the antilipolytic hormone insulin. However, much still remains to be learned about regulation of lipolysis by intracellular signaling pathways in adipocytes. Here we show that galectin-12, a member of a β-galactoside-binding lectin family preferentially expressed by adipocytes, functions as an intrinsic negative regulator of lipolysis. Galectin-12 is primarily localized on lipid droplets and regulates lipolytic protein kinase A signaling by acting upstream of phosphodiesterase activity to control cAMP levels. Ablation of galectin-12 in mice results in increased adipocyte mitochondrial respiration, reduced adiposity, and ameliorated insulin resistance/glucose intolerance. This study identifies unique properties of this intracellular galectin that is localized to an organelle and performs a critical function in lipid metabolism. These findings add to the significant functions exhibited by intracellular galectins, and have important therapeutic implications for human metabolic disorders.

Successful production of genetically modified mouse lines is dependent on germline transmission (GLT) of mutant alleles from chimeras. When natural mating fails to achieve GLT due to male infertility, sickness, or other problems, sperm can be harvested from chimeras and used for assisted reproductive technologies such as in vitro fertilization (IVF) to attempt to "rescue" GLT. However, a rational, evidence-based approach to determine if such extraordinary efforts should be attempted on a chimera has not been established. Therefore, in the present study we assessed the production, quality and genotype of epididymal sperm harvested from male chimeras generated by blastocyst or morula microinjection of gene targeted embryonic stem (ES) cell clones containing a LacZ expression cassette and that failed to achieve GLT. Results of this analysis enabled us to determine the cause of GLT failure, correlate coat color chimerism with the proportion of LacZ-positive sperm, and test the likelihood of achieving GLT by IVF. In 415 chimeras, 332 (80%) produced no offspring by natural mating ("infertile"), while 83 (20%) produced only wildtype offspring ("fertile"). Of the 332 infertile chimeras, 209 (63%) failed to produce any sperm whatsoever, 48 (15%) had extremely poor quality sperm, and 75 (23%) had good quality sperm. These results indicate that most chimeras that do not achieve GLT by natural mating are infertile, and the primary cause of infertility is failed spermatogenesis. Genotyping of sperm from 519 chimeras revealed a significant positive linear correlation between coat color chimerism and mean percentage of LacZ-positive sperm (R(2) = 0.95). Finally, IVF using good quality, LacZ-positive sperm from fertile and infertile chimeras "rescued" GLT for 19 out of 56 genes. We conclude that an assessment of coat color chimerism together with sperm quality and genotype can better inform the selection of chimeras for IVF to rescue GLT than coat color chimerism alone.

The sustained-release formulation of meloxicam (MSR) is a compounded NSAID that may provide pain relief for as long as 72 h after administration. MSR injection-site skin reactions have occurred in several species but have not previously been observed in mice. We investigated the development and progression of localized skin reactions after a single injection of MSR in Crl:CD1(ICR), C57BL/6J, and BALB/cJ mice. Each mouse received a subcutaneous injection of MSR (n = 60), standard-formulation meloxicam (MEL; n = 24) or saline (control; SC; n = 24) and was scored daily according to a 5-point system for erythema and mass characteristics. Mice were euthanized at either 7 or 14 d after injection and underwent postmortem analysis. MSR-treated mice had more erythematous and mass reactions than did MEL and SC mice. Mass lesions developed in 49 MSR mice (82%; 95% CI, 70% to 90%), 5 MEL animals (21%; 95% CI, 7% to 42%), and 1 SC mouse. MSR-treated BALB/cJ developed erythematous lesions less frequently than similarly treated Crl:CD1(ICR) or C57BL/6J. Lesions often were ventrolateral to the injection site. The median times to the appearance of mass and erythematous lesions were 2 d and 3 d, respectively. Histologically, the erythematous and mass reactions correlated with necrotizing to pyogranulomatous injection-site panniculitis. Inflammation severity scores at 7 and 14 d after injection were greater in the MSR-treated group than the other 2 groups. No strain- or sex-associated differences emerged except that inflammation severity scores at day 14 were higher in Crl:CD1(ICR) females than males. The character of the inflammatory response in MSR-treated mice did not differ between 7 and 14 d after injection, indicating that MSR-induced inflammation is slow to resolve. The ventral migration and delayed onset of MSR injection-site reactions could result in their being attributed to another cause or not being identified. Researchers and clinicians should be aware of the potential for slowly resolving injection-site reactions with MSR.

Background

Modification of cryoprotective medium (CPM) R18S3 (18% raffinose and 3% skim milk) by addition of monothioglycerol (MTG) or L-glutamine (Glu) has been shown to improve in vitro fertilization (IVF) using mouse sperm cryopreserved in cryostraws. However, whether these CPMs can be applied effectively to sperm cryopreserved in cryovials is unknown.

Objective

The study was to determine the comparative effectiveness of using R18S3, R18S3+Glu (100mM and 87 mM), or R18S3+MTG (477 µM) to cryopreserve various sample volumes of mouse sperm in cryovials and cryostraws.

Methods

This study compared the effects of different CPMs on motility of fresh and frozen-thawed C57BL/6J sperm and on IVF rate of C57BL/6J sperm cryopreserved in different CPMs and containers with different volumes, and then used technologies developed to cryopreserve and recover sperm of knockout mouse lines on inbred C57BL/6 backgrounds.

Results

Glutamine at 100 mM inhibited, but MTG at 477 µM protected, fresh sperm motility significantly (P < 0.05). Sperm cryopreserved in R18S3+MTG had significantly better (P < 0.05) post-thaw progressive motility and IVF rate than when cryopreserved in R18S3 alone, R18S3+Glu (100 mM), or RSGlu87 (15.7% raffinose, 2.6% skim milk, and 87 mM L-glutamine). There was no significant difference in IVF rates among sperm cryopreserved with R18S3+MTG in cryovials or in cryostraws (P > 0.05). Sperm from 63 knockout mouse lines on C57BL/6 backgrounds cryopreserved using R18S3+MTG in cryovials were all recovered successfully to genotypically-confirmed offspring.

Conclusion

Mouse sperm on C56BL/6 backgrounds can be successfully cryopreserved in cryovials using R18S3+MTG.

Marker assisted speed congenics technique is commonly used to facilitate backcrossing of mouse strains in nearly half the time it normally takes otherwise. Traditionally, the technique is performed by analyzing PCR amplified regions of simple sequence length polymorphism (SSLP) markers between the recipient and donor strains: offspring with the highest number of markers showing the recipient genome across all chromosomes is chosen for the next generation. Although there are well-defined panels of SSLP makers established between certain pairs of mice strains, they are incomplete for most strains. The availability of well-established marker sets for speed congenic screens would enable the scientific community to transfer mutations across strain backgrounds. In this study, we tested the suitability of over 400 SSLP marker sets among 10 mouse strains commonly used for generating genetically engineered models. The panel of markers presented here can readily identify the specified strains and will be quite useful in marker assisted speed congenic screens. Moreover, unlike newer single nucleotide polymorphism (SNP) array methods which require sophisticated equipment, the SSLP markers panel described here only uses PCR and agarose gel electrophoresis of amplified products; therefore it can be performed in most research laboratories.