Ri-ichi Takahashi

Transposon-tagged mutagenesis in the rat.

Although the laboratory rat (Rattus norvegicus) is an indispensable experimental animal for biomedical research and drug development, the lack of embryonic stem cell lines hampers gene-knockout studies. Here we report the successful generation of insertional mutant rats using the Sleeping Beauty (SB) transposon system. This would benefit a variety of biomedical research fields for which the rat model is better suited than the mouse model.

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-ChrXMSM, 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-ChrXMSM 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-ChrXMSM 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-ChrXMSM spermatozoa have a defect in penetration through the zona pellucida of eggs.

Chemically Induced Forestomach Papillomas in Transgenic Mice Carry Mutant Human c-Ha-ras Transgenes

Forestomach papillomas and skin papillomas were induced very efficiently by a single dose administration of the chemical carcinogen methylnitrosourea (MNU) in transgenic mice (rasH2 line) carrying human hybrid c-Ha-ras genes, which encode the prototype p21 gene product. The incidence of forestomach papillomas was dose dependent; when 50 mg/kg of MNU were administered i.p., all of the transgenic mice (56 of 56) developed forestomach papillomas within 12 weeks after administration, whereas 5 and 0.5 mg/kg of MNU induced papillomas in 2 of 19 and 1 of 19 mice, respectively. Nine of 56 transgenic mice (16%) also developed skin papillomas at sites wounded by bites or scratches. Only 1 of 77 nontransgenic littermates developed forestomach papillomas after administration of 50 mg/kg of MNU, and no skin papillomas appeared within 12 weeks after MNU administration. The transgenes (integrated copy number, 5-6) in the tumors developed in 55 of 56 affected transgenic mice (98%) contained at least 1 copy of the transgene that was activated by somatic point mutation at the 12th codon, from GGC (Gly) to GAC (Asp). Because somatic point mutations at the 12th or 61st codon of transgenes have never been detected in normal tissues of transgenic mice thus far examined, these mutational activations of transgenes are tumor-specific events. RNA expression of these activated transgenes was also detected. From these results, it is suggested that somatic mutational activation of the human c-Ha-ras transgene plays a causative role in the occurrence of forestomach and skin papillomas induced by MNU administration in these transgenic mice. This transgenic mouse provides a unique screening system for chemicals that induce or suppress papillomagenesis.

Position‐independent and high‐level expression of human α‐lactalbumin in the milk of transgenic rats carrying a 210‐kb YAC DNA

The level of expression of transgenes in transgenic animals varies among lines, and is often much lower than that of endogenous genes (position effects). In order to surmount position effects and establish a more efficient production system of transgenic animals producing pharmaceutical proteins in their milk, transgenic rats carrying 210‐kb YAC DNA containing the human α‐lactalbumin gene were produced. Three transgenic lines transmitted the transgene to the next generation. They had one copy of the α‐lactalbumin gene and secreted human α‐lactalbumin in their milk at concentrations of 2.0–4.3 mg/ml. No position effect was seen. The transgene was expressed specifically in the mammary gland of the transgenic rats. The 210‐kb region is thought to contain all the DNA elements required for proper expression of the human α‐lactalbumin gene. The YAC carrying the human α‐lactalbumin gene is a potential vector for the expression of foreign genes in the mammary gland. Mol. Reprod. Dev. 47:157–163, 1997.

Production of transgenic rats using cryopreserved pronuclear‐stage zygotes

We investigated the application of cryopreserved pronuclear‐stage zygotes for the production of transgenic rats. Most of the pronuclear‐stage zygotes cryopreserved by conventional two‐step freezing or vitrification appeared morphologically normal, but the proportion of frozen zygotes that developed into fetuses following transfer (59.7–60.2%) was higher than that of vitrified zygotes (5.5–22.1%). When the frozen‐thawed zygotes were used for DNA microinjection, 97.5% survived after DNA microinjection and 25.1% of the transferred zygotes developed into fetuses. These proportions were comparable to those of the fresh control zygotes (97.0% and 30.0%, respectively). The integration efficiency of the exogenous DNA into fetuses was similar between the frozen group (3.3% per injected zygote) and the control group (3.5%). These results indicate that pronuclear‐stage rat zygotes can be successfully cryopreserved by conventional two‐step freezing for production of transgenic rats.

Establishment of lacZ-transgenic rats: a tool for regenerative research in myocardium.

Animals transgenic (Tg) for reporter genes would be useful to following a given cell lineage during differentiation and regeneration processes. Here, we established a beta-galactosidase (lacZ) Tg rat to use as a tool for regenerative research. Strong lacZ expression was observed in the skeletal muscles, myocardium, pancreas, and skin obtained from these lacZ-Tg rats, and moderate lacZ expression was observed in the liver, spleen, kidney, and cartilage. In contrast, brain, vessels, lung, adrenal gland, small intestine, blood leukocytes, bone marrow (BM) cells, and peripheral blood cells showed no lacZ expression. To test whether this lacZ-Tg rat could be used for regenerative research in myocardium, we induced myocardial injury after a lacZ-Tg BM transplant (BMT) into wild-type rats. The results show that lacZ-positive cardiomyocytes were found in the peri-infarct and uninjured myocardium in the BMT recipient rats. These findings suggest that lacZ-Tg rats are useful tool for regenerative research in the myocardium.

Overexpression of c-myc induces apoptosis at the prophase of meiosis of rat primary spermatocytes

Transgenic rats expressing the rat c‐myc gene under the control of the human metallothionein II A promoter were produced. We found that the female transgenic rats were fertile, but that the male transgenic rats were sterile. Atrophy of the seminiferous tubules and depletion of sperm were observed in the sterile male testes. The expression of differential stage‐specific mRNAs, including those of the c‐kit receptor proto‐oncogene, meiotic heat‐shock protein 70 gene, acrosin gene, and transition protein 1 gene, was analyzed by the reverse transcriptase‐polymerase chain reaction during spermatogenesis. The results suggested that spermatogenesis in these sterile rats were arrested at the prophase of meiosis in the primary spermatocytes. We found that apoptotic DNA fragmentation occured in primary spermatocytes of the sterile transgenic rats. These results suggest that overexpression of the c‐myc gene induces apoptosis at the prophase meiosis of the primary spermatocytes thereby causing male sterility in the c‐myc transgenic rats.

High-level expressing YAC vector for transgenic animal bioreactors.

The position effect is one major problem in the production of transgenic animals as mammary gland bioreactors. In the present study, we introduced the human growth hormone (hGH) gene into 210‐kb human α‐lactalbumin position‐independent YAC vectors using homologous recombination and produced transgenic rats via microinjection of YAC DNA into rat embryos. The efficiency of producing transgenic rats with the YAC vector DNA was the same as that using plasmid constructs. All analyzed transgenic rats had one copy of the transgene and produced milk containing a high level of hGH (0.25–8.9 mg/ml). In transgenic rats with the YAC vector in which the human α‐lactalbumin gene was replaced with the hGH gene, tissue specificity of hGH mRNA was the same as that of the endogenous rat α‐lactalbumin gene. Thus, the 210‐kb human α‐lactalbumin YAC is a useful vector for high‐level expression of foreign genes in the milk of transgenic animals. Mol. Reprod. Dev. 52:414–420, 1999.

Differentiation Stage-Specific Requirement in Hypoxia-Inducible Factor-1α–Regulated Glycolytic Pathway during Murine B Cell Development in Bone Marrow

Hypoxia-inducible factor (HIF)-1α plays a central role in oxygen homeostasis and energy supply by glycolysis in many cell types. We previously reported that an HIF-1α gene deficiency caused abnormal B cell development and autoimmunity. In this study we show that HIF-1α–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-1α–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-1α–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-1α–deficient cells than in wild-type cells. In agreement with these findings, HIF-1α–deficient cells used pyruvate more efficiently than wild-type cells. The key role of HIF-1α–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-1α plays an important role in B cell development.

Analysis of control elements for position-independent expression of human α-lactalbumin YAC

A major problem in the production of transgenic animal bioreactors using microinjections is the low production rate of high‐expressing transgenic animals due to the position effect. We previously reported that transgenic rats carrying the 210 kb yeast artificial chromosome (YAC) including the human α‐lactalbumin gene express the transgene in a position‐independent manner. The 210 kb YAC was thought to have all the elements necessary for position‐independent expression. In this paper, we constructed fragmented YAC clones and a cosmid clone, and produced transgenic rats to analyze these elements. Transgenic rats with both the 50 kb upstream and downstream regions of the α‐lactalbumin gene had position‐independent expression. Transgenic rats with the 20 kb upstream and downstream regions, however, had position‐dependent expression. Therefore, all the elements necessary for position‐independent expression are thought to be located in the 50 kb upstream to 50 kb downstream region of the α‐lactalbumin gene. Furthermore, we replaced the human α‐lactalbumin promoter with the bovine αS1‐casein promoter in the 210 kb YAC and produced transgenic rats. Position‐dependent expression was observed. The elements required for position‐independent expression of the bovine αS1‐casein gene are different from those required for the human α‐lactalbumin gene, despite the fact that the two genes have the same tissue and developmental specificity. Mol. Reprod. Dev. 54:17–23, 1999.