Necessity of integrated genomic analysis to establish a designed knock-in mouse from CRISPR-Cas9-induced mutants.

The CRISPR-Cas9 method for generation of knock-in mutations in rodent embryos yields many F0 generation candidates that may have the designed mutations. The first task for selection of promising F0 generations is to analyze genomic DNA which likely contains a mixture of designed and unexpected mutations. In our study, while generating Prlhr-Venus knock-in reporter mice, we found that genomic rearrangements near the targeted knock-in allele, tandem multicopies at a target allele locus, and mosaic genotypes for two different knock-in alleles occurred in addition to the designed knock-in mutation in the F0 generation. Conventional PCR and genomic sequencing were not able to detect mosaicism nor discriminate between the designed one-copy knock-in mutant and a multicopy-inserted mutant. However, by using a combination of Southern blotting and the next-generation sequencing-based RAISING method, these mutants were successfully detected in the F0 generation. In the F1 and F2 generations, droplet digital PCR assisted in establishing the strain, although a multicopy was falsely detected as one copy by analysis of the F0 generation. Thus, the combination of these methods allowed us to select promising F0 generations and facilitated establishment of the designed strain. We emphasize that focusing only on positive evidence of knock-in can lead to erroneous selection of undesirable strains.

Differentiation stage-specific requirement in hypoxia-inducible factor-1alpha-regulated glycolytic pathway during murine B cell development in bone marrow.

Hypoxia-inducible factor (HIF)-1alpha plays a central role in oxygen homeostasis and energy supply by glycolysis in many cell types. We previously reported that an HIF-1alpha gene deficiency caused abnormal B cell development and autoimmunity. In this study we show that HIF-1alpha-enabled glycolysis during B cell development is required in a developmental stage-specific manner. Supporting this conclusion are observations that the glycolytic pathway in HIF-1alpha-deficient B220(+) bone marrow cells is much less functionally effective than in wild-type control cells. The expression of genes encoding the glucose transporters and the key glycolytic enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bishosphatase 3, was greatly reduced in HIF-1alpha-deficient cells. The compensatory adaptation to the defect of glycolysis was reflected in higher levels of expression of respiratory chain-related genes and TCA cycle-related genes in HIF-1alpha-deficient cells than in wild-type cells. In agreement with these findings, HIF-1alpha-deficient cells used pyruvate more efficiently than wild-type cells. The key role of HIF-1alpha-enabled glycolysis in bone marrow B cells was also demonstrated by glucose deprivation during in vitro bone marrow cell culture and by using a glycolysis inhibitor in the bone marrow cell culture. Taken together, these findings indicate that glucose dependency differs at different B cell developmental stages and that HIF-1alpha plays an important role in B cell development.

The endoplasmic reticulum stress-inducible protein Niban regulates eIF2alpha and S6K1/4E-BP1 phosphorylation.

The Niban/NIBAN gene is specifically expressed in hereditary renal carcinomas of model animals and in human malignancies, including renal cancers. Although the expression profiles of Niban/NIBAN suggest that it plays an important role in carcinogenesis, no functional information has yet been reported. In this study, we found that the levels of Niban/NIBAN mRNA and protein were induced by treatment with tunicamycin, an inducer of endoplasmic reticulum (ER) stress. To elucidate Niban's in vivo function, we generated a Niban knockout mouse. Niban(-/-) mouse showed no obvious phenotype. Unexpectedly, we found that eukaryotic translational initiation factor (eIF) 2alpha phosphorylation, which is up-regulated during ER stress, was increased in Niban(-/-) cells relative to wild-type control cells. In addition, decreased phosphorylation of p70 ribosomal S6 subunit kinase (S6K) 1 and eukaryotic initiation factor 4E-binding protein (4E-BP) 1 was also detected in Niban(-/-) cells. Similar effects were observed following transfection of NIBAN-specific interfering RNAs in HeLa cells. Thus, Niban positively affects protein translation machineries. Additionally, suppression of NIBAN expression in HeLa cells promoted apoptosis. Together these results suggest that Niban is involved in the ER stress response, and that Niban can modulate cell death signaling by regulating translation.

Production of progeny mice by intracytoplasmic sperm injection of repeatedly frozen and thawed spermatozoa experimental technique.

Methods for generating genetically engineered mice have progressed, and the number of valuable mouse strains has increased rapidly, requiring methods for managing and maintaining these strains. Sperm cryopreservation and assisted-reproduction techniques, such as intracytoplasmic sperm injection (ICSI), can contribute greatly; however, the number of possible progeny is limited due to the finite number of cryopreserved preparations. The ability to refreeze and reuse sperm preparations would extend the utility of each cryopreserved sperm preparation. The purpose of this study was to develop a reproduction protocol involving ICSI that yielded live progeny after repeated freezing and thawing of a cryopreserved sperm preparation. We used mouse sperm subjected to repeat freezing and thawing in TYH medium for in vitro fertilization. Three inbred strains of laboratory mice--C57BL/6J, BALB/cA, and C3H/HeN--were reproduced by ICSI after reuse of a sperm preparation that had been frozen and thawed repeatedly. In particular, C57BL/6J progeny could be reproduced from spermatozoa frozen and thawed 10 times. From these results, we conclude that the reuse of cryopreserved spermatozoa can extend the opportunities for reproduction of progeny from cryopreserved sperm and can increase the utility of cryopreserved preparations as bioresources. Our results broaden management options regarding bioresource banking, particularly for mice.

Disruption of genetic interaction between two autosomal regions and the X chromosome causes reproductive isolation between mouse strains derived from different subspecies.

Reproductive isolation that initiates speciation is likely caused by incompatibility among multiple loci in organisms belonging to genetically diverging populations. Laboratory C57BL/6J mice, which predominantly originated from Mus musculus domesticus, and a MSM/Ms strain derived from Japanese wild mice (M. m. molossinus, genetically close to M. m. musculus) are reproductively isolated. Their F1 hybrids are fertile, but successive intercrosses result in sterility. A consomic strain, C57BL/6J-ChrX(MSM), which carries the X chromosome of MSM/Ms in the C57BL/6J background, shows male sterility, suggesting a genetic incompatibility of the MSM/Ms X chromosome and other C57BL/6J chromosome(s). In this study, we conducted genomewide linkage analysis and subsequent QTL analysis using the sperm shape anomaly that is the major cause of the sterility of the C57BL/6J-ChrX(MSM) males. These analyses successfully detected significant QTL on chromosomes 1 and 11 that interact with the X chromosome. The introduction of MSM/Ms chromosomes 1 and 11 into the C57BL/6J-ChrX(MSM) background failed to restore the sperm-head shape, but did partially restore fertility. This result suggests that this genetic interaction may play a crucial role in the reproductive isolation between the two strains. A detailed analysis of the male sterility by intracytoplasmic sperm injection and zona-free in vitro fertilization demonstrated that the C57BL/6J-ChrX(MSM) spermatozoa have a defect in penetration through the zona pellucida of eggs.

Physical performance and soleus muscle fiber composition in wild-derived and laboratory inbred mouse strains.

We compared four inbred mouse strains in their physical performance, measured as a maximal treadmill running time, characteristics of soleus muscle, anatomic character, and growth. The strains used were Mus musculus domesticus [C57BL/6 (B6) and BALB/c], Mus musculus molossinus (MSM/Ms), and Mus spretus. Maximal running time was significantly different among these four mouse strains. Running time until exhaustion was highest in MSM/Ms and lowest in M. spretus. Maximal times for the laboratory mouse strains were nearly identical. Soleus muscle fiber type and cross-sectional area also differed significantly among the species. In particular, M. spretus was significantly different from the other inbred mouse strains. Growth in the wild-derived inbred mice appeared to be complete earlier than in the laboratory mice, and the body size of the wild strains was about half that of the laboratory strains. From these results, we propose that wild-derived inbred mouse strains are useful models for enhancing phenotypic variation in physical performance and adaptability.