lacZ Transgenic Mice Research Articles

Analysis of Biomarkers of DNA Damage and Mutagenicity in Mice Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in the mouse via unresolved mechanisms. For this report, complementary and previously described methods were used to assess in vivo genotoxicity and/or mutagenicity of ACN in several mouse models, including (i) female mice devoid of cytochrome P450 2E1 (CYP2E1), which yields the epoxide intermediate cyanoethylene oxide (CEO), (ii) male lacZ transgenic mice, and (iii) female (wild-type) B6C3F1 mice. Exposures of wild-type mice and CYP2E1-null mice to ACN at 0, 2.5 (wild-type mice only), 10, 20, or 60 (CYP2E1-null mice only) mg/kg body weight by gavage for 6 weeks (5 days/week) produced no elevations in the frequencies of micronucleated erythrocytes, but induced significant dose-dependent increases in DNA damage, detected by the alkaline (pH >13) Comet assay, in one target tissue (forestomach) and one nontarget tissue (liver) of wild-type mice only. ACN exposures by gavage also caused significant dose-related elevations in the frequencies of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) reporter gene of T-lymphocytes from spleens of wild-type mice; however, Hprt mutant frequencies were significantly increased in CYP2E1-null mice only at a high dose of ACN (60 mg/kg) that is lethal to wild-type mice. Similarly, drinking water exposures of lacZ transgenic mice to 0, 100, 500, or 750 ppm ACN for 4 weeks caused significant dose-dependent elevations in Hprt mutant frequencies in splenic T-cells; however, these ACN exposures did not increase the frequency of lacZ transgene mutations above spontaneous background levels in several tissues from the same animals. Together, the Comet assay and Hprt mutant frequency data from these studies indicate that oxidative metabolism of ACN by CYP2E1 to CEO is central to the induction of the majority of DNA damage and mutations in ACN-exposed mice, but ACN itself also may contribute to the carcinogenic modes of action via mechanisms involving direct and/or indirect DNA reactivity.

Radiation sensitivity of the gastrula-stage embryo: Chromosome aberrations and mutation induction in lacZ transgenic mice: The roles of DNA double-strand break repair systems

At the gastrula phase of development, just after the onset of implantation, the embryo proper is characterized by extremely rapid cell proliferation. The importance of DNA repair is illustrated by embryonic lethality at this stage after ablation of the genes involved. Insight into mutation induction is called for by the fact that women often do not realize they are pregnant, shortly after implantation, a circumstance which may have important consequences when women are subjected to medical imaging using ionizing radiation. We screened gastrula embryos for DNA synthesis, nuclear morphology, growth, and chromosome aberrations (CA) shortly after irradiation with doses up to 2.5Gy. In order to obtain an insight into the importance of DNA repair for CA induction, we included mutants for the non-homologous end joining (NHEJ) and homologous recombination repair (HRR) pathways, as well as Parp1-/- and p53+/− embryos. With the pUR288 shuttle vector assay, we determined the radiation sensitivity for point mutations and small deletions detected in young adults. We found increased numbers of abnormal nuclei 5h after irradiation; an indication of disturbed development was also observed around this time. Chromosome aberrations 7h after irradiation arose in all genotypes and were mainly of the chromatid type, in agreement with a cell cycle dominated by S-phase. Increased frequencies of CA were found for NHEJ and HR mutants. Gastrula embryos are unusual in that they are low in exchange induction, even after compromised HR. Gastrula embryos were radiation sensitive in the pUR288 shuttle vector assay, giving the highest mutation induction ever reported for this genetic toxicology model. On theoretical grounds, a delayed radiation response must be involved. The compromised developmental profile after doses up to 2.5Gy likely is caused by both apoptosis and later cell death due to large deletions. Our data indicate a distinct radiation-sensitive profile of gastrula embryos, including some stage-specific aspects that are not as yet understood.

Solar-UV-signature mutation prefers TCG to CCG: extrapolative consideration from UVA1-induced mutation spectra in mouse skin

UVA1 exerts its genotoxicity on mammalian skin by producing cyclobutane pyrimidine dimers (CPDs) in DNA and preferentially inducing solar-UV-signature mutations, C → T base substitution mutations at methylated CpG-associated dipyrimidine (Py-mCpG) sites, as demonstrated previously using a 364 nm laser as a UVA1 source and lacZ-transgenic mice that utilize the transgene as a mutational reporter. In the present study, we confirmed that a broadband UVA1 source induced the same mutation profiles in mouse epidermis as the UVA1 laser, generalizing the previous result from a single 364 nm to a wider wavelength range of UVA1 (340-400 nm). Combined with our previous data on the mutation spectra induced in mouse epidermis by UVB, UVA2 and solar UVR, we proved that the solar-UV-signature mutation is commonly observed in the wavelength range from UVB to UVA, and found that UVA1 induces this mutation more preferentially than the other shorter wavelength ranges. This finding indicates that the solar-UV-signature mutation-causing CPDs, which are known to prefer Py-mCpG sites, could be produced with the energy provided by the longer wavelength region of UVR, suggesting a photochemical reaction through the excitation of pyrimidine bases to energy states that can be accomplished by absorption of even low-energy UVR. On the other hand, the lower proportions of solar-UV-signature mutations observed in the mutation spectra for UVB and solar UVR indicate that the direct photochemical reaction through excited singlet state of pyrimidine bases, which can be accomplished only by high-energy UVR, is also involved in the mutation induction at those shorter wavelengths of UVR. We also found that the solar-UV signature prefers 5'-TCG-3' to 5'-CCG-3' as mutational target sites, consistent with the fact that UVA induces CPDs selectively at thymine-containing dipyrimidine sites and that solar UVR induces them preferably at Py-mCpG sites. However, the mutation spectrum in human p53 gene from non-melanoma skin cancers shows the opposite preference for 5'-CCG-3' sites. This apparent discrepancy in the site preference seems to result from the lack of 5'-TCG-3' sites mutable to missense mutations on the nontranscribed strand of human p53 gene, which should be evolutionally acquired under selective pressure from the sun.

Action Spectrum Analysis of UVR Genotoxicity for Skin: The Border Wavelengths between UVA and UVB Can Bring Serious Mutation Loads to Skin

UVR causes erythema, which has been used as a standardized index to evaluate the risk of UVR for human skin. However, the genotoxic significance of erythema has not been elucidated clearly. Here, we characterized the wavelength dependence of the genotoxic and erythematic effects of UVR for the skin by analyzing the induction kinetics of mutation and inflammation in mouse skin using lacZ-transgenic mice and monochromatic UVR sources. We determined their action spectra and found a close correlation between erythema and an epidermis-specific antigenotoxic response, mutation induction suppression (MIS), which suppressed the mutant frequencies (MFs) to a constant plateau level only 2-3-fold higher than the background MF at the cost of apoptotic cell death, suggesting that erythema may represent the threshold beyond which the antigenotoxic but tissue-destructive MIS response commences. However, we unexpectedly found that MIS attenuates remarkably at the border wavelengths between UVA and UVB around 315 nm, elevating the MF plateaus up to levels ∼40-fold higher than the background level. Thus, these border wavelengths can bring heavier mutation loads to the skin than the otherwise more mutagenic and erythematic shorter wavelengths, suggesting that erythema-based UVR risk evaluation should be reconsidered.

Osteoclasts in Multiple Myeloma Are Derived from Gr-1+CD11b+Myeloid-Derived Suppressor Cells

Osteoclasts play a key role in the development of cancer-associated osteolytic lesions. The number and activity of osteoclasts are often enhanced by tumors. However, the origin of osteoclasts is unknown. Myeloid-derived suppressor cells (MDSCs) are one of the pre-metastatic niche components that are induced to expand by tumor cells. Here we show that the MDSCs can differentiate into mature and functional osteoclasts in vitro and in vivo. Inoculation of 5TGM1-GFP myeloma cells into C57BL6/KaLwRij mice led to a significant expansion of MDSCs in blood, spleen, and bone marrow over time. When grown in osteoclastogenic media in vitro, MDSCs from tumor-challenged mice displayed 14 times greater potential to differentiate into mature and functional osteoclasts than those from non-tumor controls. Importantly, MDSCs from tumor-challenged LacZ transgenic mice differentiated into LacZ+osteoclasts in vivo. Furthermore, a significant increase in tumor burden and bone loss accompanied by increased number of osteoclasts was observed in mice co-inoculated with tumor-challenged MDSCs and 5TGM1 cells compared to the control animals received 5TGM1 cells alone. Finally, treatment of MDSCs from myeloma-challenged mice with Zoledronic acid (ZA), a potent inhibitor of bone resorption, inhibited the number of osteoclasts formed in MDSC cultures and the expansion of MDSCs and bone lesions in mice. Collectively, these data provide in vitro and in vivo evidence that tumor-induced MDSCs exacerbate cancer-associated bone destruction by directly serving as osteoclast precursors.

Transition from organogenesis to stem cell maintenance in the mouse adrenal cortex

Mice showing mosaic expression of an appropriate marker gene that is activated during development provide simple tools for investigating cell lineages. We used the mosaic β-galactosidase staining patterns in adrenal cortices of 21OH/ LacZ transgenic mice to study both organogenesis and maintenance of the adult tissue. Randomly orientated mosaic patterns present in embryonic day 14.5 (E14.5) adrenals changed progressively during the perinatal period from discrete spots, via patches and radial arrays, to radial stripes, which first emerged between postnatal days 0 and 7 (P0 and P7). The mosaic radial stripe pattern was fully established by P21 and remained unchanged throughout the adult period (8–52 weeks). The mouse adrenal gland grew continuously between E14.5 and P21, including the period during which stripes emerge. Ki67-positive, proliferative cells in the adrenal cortex were mainly localized to the outer cell layers between E18.5 and P3. By P10, cell proliferation had increased, and the proliferative region had expanded but was still mainly confined to the outer cortex. Correlation of changes in mosaic patterns in 21OH/LacZ adrenal cortices with the locations of adrenocortical cell proliferation suggest that the radial stripes arise by edge-biased growth during the perinatal period, even if they are maintained by stem cells in adults. The stability of the adult stripe pattern suggests that stem cell function is unchanged between 8 and 52 weeks.

Mononuclear cell therapy attenuates atherosclerosis in apoE KO mice

BackgroundRecent studies have highlighted the potential of cell therapy for atherosclerosis. The aim of this study was to evaluate the effects of mononuclear cell (MNC) therapy on the development of atherosclerotic lesions in the apolipoprotein E knockout (apoE KO) mouse.MethodsWe investigated vascular lipid deposition, vascular remodeling, oxidative stress, and endothelial nitric oxide synthase (eNOS) expression in apoE KO mice treated with spleen MNCs isolated from lacZ transgenic mice (apoE KO-MNC) for 8 weeks compared to untreated control mice (apoE KO).ResultsHistological analysis of aortas showed a significant reduction in the lipid deposition area in apoE KO-MNC mice compared to apoE KO mice (0.051 ± 0.004 vs 0.117 ± 0.016 mm2, respectively, p < 0.01). In addition, vessel morphometry revealed that MNC therapy prevented the outward (positive) remodeling in apoE KO mice that is normally observed (apoE KO-MNC: 0.98 ± 0.07 vs apoE KO: 1.37 ± 0.09), using wild-type mice (C57BL/6J) as a reference. ApoE KO-MNC mice also have reduced production of superoxide anions and increased eNOS expression compared to apoE KO mice. Finally, immunohistochemistry analysis revealed a homing of endothelial progenitor cells (EPCs) in the aortas of apoE KO-MNC mice.ConclusionMNC therapy attenuates the progression of atherosclerosis in the aortas of apoE KO mice. Our data provide evidence that the mechanism by which this attenuation occurs includes the homing of EPCs, a decrease in oxidative stress and an upregulation of eNOS expression.

Effects of potential dietary inhibitors of endogenous DNA damage on mutagenesis and lipid peroxidation in lacZ mice

The effects of a nine month administration of dietary: (1) 3H-1,2-dithiole-3-thione (D3T), (2) N-acetylcysteine (NAC), (3) antioxidant vitamin mix, (vitamin C+E), (4) free radical scavenger, amifostine, and (5) calorie restriction, (CR), on mutagenesis and lipid peroxidation in lung, kidney, spleen and liver of lacZ transgenic mice were examined. These agents/diets were chosen because they might inhibit certain proposed mechanisms of endogenous damage to DNA. The agents were added to a high fat, reduced antioxidant AIN-76 diet, to better approximate a Western style diet than the conventional AIN-76 diet. As the lacZ gene is not expressed, mutations in that gene are neutral, and simply accumulate over time. The mutant fractions in control mice increased about 50-100%. Most of the agents inhibited to various extents the age-related increase in mutagenesis in lung, kidney, and/or spleen, but no inhibition was observed in liver. There was no significant effect of age on lipid peroxidation levels in controls, possibly reflecting steady state turnover of lipid peroxidation products. Almost all of the treatments except D3T inhibited lipid peroxidation in most organs to different degrees. The vitamin C+E mix was the most effective at inhibiting lipid peroxidation, but a single most effective inhibitor of mutagenesis could not be discerned. Some associations were observed between the reduction in lipid peroxidation and the inhibition of mutagenesis. The results are consistent with a partial role for oxidative stress in the age-related increase in mutagenesis. These observations may have implications for chemoprevention of carcinogenesis.

Effects of calorie restriction on the age-dependent accumulation of mutations in the small intestine of lacZ-transgenic mice

To understand the effect of calorie restriction on genome maintenance systems, the age-dependent accumulation of mutations in animals maintained on high and low calorie diets was examined using lacZ-transgenic mice. Mice were fed a diet of 95kcal/w or 65kcal/w from 2 to 17 months of age. The mutation frequencies in the lacZ gene in epithelial tissues from the small intestine were examined at 12 and 17 months. Mutation frequencies were found to be lower in mice fed with a low calorie diet than in mice fed with a high calorie diet at the two age points. The molecular nature of the mutations was examined with DNA sequencing. It showed a predominance of transversions from G:C to T:A, and this is a typical type of mutation induced by reactive oxygen species. The fraction of this type of mutation among the different types of mutations detected was not affected by calorie restriction. The percentage of the other types of mutation was not influenced either. These results suggest that calorie restriction reduces the age-dependent accumulation of mutations by stimulating or inducing various types of DNA protection and repair systems rather than protecting cells against any specific type of DNA alteration.

Poly (ADP‐ribose) polymerase‐1 deficiency does not affect ethylnitrosourea mutagenicity in liver and testis of lacZ transgenic mice

Poly (ADP-ribose) polymerase-1 (Parp1) has been implicated in DNA base excision repair, single- and double-strand break repair pathways, as well as in cell death by apoptosis or necrosis. We used Parp1(-/-) lacZ plasmid-based transgenic mice to investigate whether Parp1 deficiency influences the in vivo mutagenic and clastogenic response to the alkylating agent N-ethyl-N-Nitrosourea (ENU) in somatic and germ-cell tissues. The comparison of the lacZ mutant frequencies (MFs) between Parp1(+/+) and Parp1(-/-) mice showed that the ablation of Parp1 does not affect the spontaneous or ENU-induced MFs in liver and testis. In addition, the spectrum of the ENU-induced mutations was not dependent on the Parp1 status, given that similar spectra, consisting mostly of point mutations and a small fraction of deletions/insertions, wereobserved in organs of both Parp1(-/-) and Parp1(+/+) mice. Sequencing of point mutations revealed a consistent significant increase in A:T --> T:A base substitutions, typically induced by ENU. Overall, we observed that neither the frequency nor the spectrum of ENU-induced mutations demonstrated a specificity that could be attributed to the Parp1 impairment in mice organs. The analysis of micronucleus frequency in peripheral blood reticulocytes showed that ENU was clastogenic in both Parp1(-/-) and Parp1(+/+) mice and had a strong cytotoxic effect in Parp1(-/-) mice only. The present data suggest that, at a whole-organism level, Parp1-independent repair mechanisms may be operative in the removal of ENU-induced DNA lesions or that highly damaged cells may be preferentially committed to death when Parp1 is inactivated.

Melanocyte and RPE‐specific expression in transgenic mice by mouse MART‐1/Melan‐A/mlana regulatory sequences

Figure S1. Identification and analysis of conserved non-coding sequences (CNS) 5′ to the mouse MART-1 gene. Four CNS sequences were analyzed in transient transfections and in transient transgenic embryos. Figure S2. Expression from MART-1(6.0kb)::lacZ transgenic mice. Expression is detected in embryonic eye (RPE) and in melanocytes after birth. Figure S3. MART-1 BAC targeting strategy. Table S1. Luciferase reporter plasmids. Primers and digestions used for generating the different constructs. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

In Vivo Optical Imaging of LacZ Expression Using lacZ Transgenic Mice

beta-Galactosidase (beta-gal) (encoded by the lacZ gene) has been widely used as a transgene reporter enzyme. The ability to image lacZ expression in living transgenic animals would further extend the use of this reporter. It has been reported that 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)-beta-d-galactopyranoside (DDAOG), a conjugate of beta-galactose and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one), is not only a chromogenic lacZ substrate but that the cleavage product has far-red fluorescence properties detectable by in vivo imaging. In an attempt to noninvasively image lacZ expression in vivo, we applied fluorescence imaging to a G protein-coupled receptor (GPR56), knockout (KO) mouse model, in which the lacZ gene is introduced in the GPR56 locus. Compared to wild-type (WT) mice, GPR56KO/LacZ mice showed three- to fourfold higher fluorescence intensity in the head area 5 min after tail-vein injection of DDAOG. beta-Gal staining in sections of whole brain showed strong lacZ expression in homozygotes, but not in WT mice, consistent with lacZ activity detected by in vivo imaging. The kidneys were also visualized with fluorescence imaging both in vivo and ex vivo, consistent with beta-gal staining findings. Our results demonstrate that fluorescence imaging can be used for in vivo real-time detection of lacZ activity by fluorescence imaging in lacZ transgenic mice. Thus, this technology can potentially be used to noninvasively image changes of certain endogenous molecules and/or molecular pathways in transgenic animals.

Mutagenesis induced by targeted alpha therapy using 213Bi-cDTPA-9.2.27 in lacZ transgenic mice

Targeted Alpha Therapy utilizes alpha-emitting radionuclides conjugated to monoclonal antibodies to allow specific irradiation of cancer cells whilst sparing normal, healthy tissues. The mutagenic potential of 213Bi conjugated to a human melanoma antigen- specific antibody (9.2.27) was examined using an in vivo transgenic mouse model containing multiple copies of a lacZ target gene in every cell, allowing the quantification and comparison of mutagenesis in different organs. Mice received an ip injection of 16.65 MBq of 213Bi-cDTPA-9.2.27, and were sacrificed at 24 h, 1 week and 4 weeks post-injection. Pharmacokinetic studies gave the absorbed and effective doses for each organ. The mutant frequency and mutant spectra were analysed for the brain, spleen and kidneys. The brain and spleen did not show significant increases in induced mutation frequencies compared to spontaneous background levels or changes in mutant spectra, these results being independent of p53 status. However, elevated mutation frequencies and persistent size change mutations were observed in the kidneys, but are not significant at the P=0.05 level. The effect of p53 status was also evident, as p53 heterozygotes displayed higher mutation frequencies than their wild-type counterparts, suggesting a reduction in the p53 gene may lead to an increased susceptibility to mutagenesis. These effects were time dependent and levels returned to those of the controls at 4 weeks post-irradiation, albeit with a predominant residue of size mutations. These effects were observed at activities very much higher than those expected for the therapy of human patients. As such, the induction of secondary cancer with the 213Bi-cDTPA- 9.2.27 alpha immunoconjugate is not expected to be a significant problem in the clinic.

Chick tooth induction revisited

Teeth have been missing from Aves for almost 100 million years. However, it is believed that the avian oral epithelium retains the molecular signaling required to induce odontogenesis, and this has been widely examined using heterospecific recombinations with mouse dental mesenchyme. It has also been argued that teeth can form from the avian oral epithelium owing to contamination of the mouse mesenchyme with mouse dental epithelial cells. To investigate the possibility of tooth formation from chick oral epithelium and the characteristics of possible chick enamel, we applied LacZ transgenic mice during heterospecific recombination and examined the further tooth formation. Transmission electron microscopy was used to identify the two tissues during development after heterospecific recombination. No mixing was detected between chick oral epithelium and mouse dental mesenchyme after 2 days, and secretory ameloblasts with Tomes' processes were observed after 1 week. Teeth were formed after 3 weeks with a single cusp pattern, possibly determined by epithelial factors, which is similar to that of the avian tooth in the late Jurassic period. These recombinant teeth were smaller than mouse molars, whereas perfect structures of both ameloblasts and enamel showed histological characteristics similar to those of mice. Together these observations consistent with previous report that odontogenesis is initially directed by species-specific mesenchymal signals interplaying with common epithelial signals.

Rosuvastatin reduces atherosclerotic lesions and promotes progenitor cell mobilisation and recruitment in apolipoprotein E knockout mice

Statins enhance incorporation of bone marrow-derived cells into experimental neointimal lesions. However, the contribution of progenitor cells to progression of spontaneous atherosclerotic plaques, and the possible modulatory role of statins in this process, remain poorly understood. We compared the effects of rosuvastatin (1 and 10mg/kg BW) and pravastatin (10mg/kg) on progenitor cell mobilisation, recruitment into atherosclerotic plaques, and lesion growth. Statins were administered over 8 weeks to apolipoprotein E knockout mice on atherogenic diet. In addition, mice were lethally irradiated, followed by transplantation of bone marrow from LacZ transgenic mice. Rosuvastatin reduced lesion area and intima-to-media ratio at the brachiocephalic artery compared to vehicle, while both parameters were not significantly altered by pravastatin. Rosuvastatin also augmented endothelialisation (P<0.05) and reduced the smooth muscle cells (SMC) content (P=0.042) of lesions. Numbers of c-kit, sca-1 and flk-1, sca-1 double-positive progenitor cells were significantly increased in rosuvastatin compared to control-treated mice, both in the bone marrow and the peripheral blood. Similarly, the number of spleen-derived acLDL, lectin double-positive progenitor cells (P=0.001) and colony-forming units (P=0.0104) was significantly increased in mice treated with rosuvastatin compared to vehicle alone. In the bone marrow, increased Akt and p42/44 MAP kinase phosphorylation and upregulated SDF1α mRNA expression were observed. Importantly, rosuvastatin treatment also increased the plasma levels of c-kit ligand (P=0.003), and the number of c-kit-positive cells within atherosclerotic lesions (P=0.041). Our findings suggest that rosuvastatin reduces the size of atherosclerotic plaques, and this effect appears to involve progenitor cell mobilisation and recruitment into vascular lesions.

Author's Response to “Donor-derived Hematopoietic Cell Contribution to Myofibers in Acid α-Glucosidase Deficiency: A Promising Progress or Back to the Beginning?”

Dear Editor, We would like to respond to the letter by Dr. Yablonka-Reuveni. This letter questioned the method we used to assay acid α-glucosidase (GAA) and therefore the interpretation of our results (Mori et al. 2008). We probed our Western blots with rabbit IgG to human urine GAA, a protein with a molecular mass of 64 kDa (Raben et al. 1998). These Western blots could not determine whether the GAA protein was contributed by donor cells that had become incorporated into the myofibers or donor cells in the interstitium. Similarly, fluorescence-activated cell sorting (FACS) analysis indicated the presence of donor cells within the muscle tissue. Additional experiments, including immunofluorescence analysis and periodic acid-Schiff (PAS) staining, were needed to determine whether the donor cells were incorporated into the myofibers or resided in the interstitium. In describing our immunofluorescence analysis, we omitted to mention that samples had been fixed with 4% paraformaldehyde in PBS for 30 min, which retains green fluorescent protein (GFP) within cells (Ojima et al. 2004). Although we corrected this mistake in our revised manuscript, this correction was not included in the exPRESS version. Of course, we should study additional cell tracking methods (e.g., male donors to female recipients or lacZ transgenic mice) to clarify that the donor cells were incorporated into myofibers. Last, we used PAS staining to confirm a reduction in glycogen storage. Because we could not estimate this reduction quantitatively, Dr. Yablonka-Reuveni suggested that cross-sections be stained with PAS, followed by staining for GFP, to show colocalization in the myofibers. Because these experiments may better show the effect of bone marrow transplantation, we intend to stain serial sections in future experiments. Myeloid cells undergo fusion with myofibers. It is not presently known, however, whether the donor cells induce expression of a missing gene on their incorporation into myofibers. The therapeutic exploitation of the fusion of donor cells with host myofibers remains an option for study. GAA knockout mice are interesting as a new cell tracking method.

Capacity of Dental Pulp Differentiation in Mouse Molars as Demonstrated by Allogenic Tooth Transplantation

Dental pulp elaborates both bone and dentin under pathological conditions such as tooth replantation/transplantation. This study aims to clarify the capability of dental pulp to elaborate bone tissue in addition to dentin by allogenic tooth transplantation using immunohistochemistry and histochemistry. After extraction of the molars of 3-week-old mice, the roots and pulp floor were resected and immediately allografted into the sublingual region in a littermate. In addition, we studied the contribution of donor and host cells to the regenerated pulp tissue using a combination of allogenic tooth transplantation and lacZ transgenic ROSA26 mice. On Days 5-7, tubular dentin formation started next to the preexisting dentin at the pulp horn where nestin-positive odontoblast-like cells were arranged. Until Day 14, bone-like tissue formation occurred in the pulp chamber, where intense tartrate-resistant acid phosphatase-positive cells appeared. Furthermore, allogenic transplantation using ROSA26 mice clearly showed that both donor and host cells differentiated into osteoblast-like cells with the assistance of osteoclast-lineage cells, whereas newly differentiated odontoblasts were exclusively derived from donor cells. These results suggest that the odontoblast and osteoblast lineage cells reside in the dental pulp and that both donor and host cells contribute to bone-like tissue formation in the regenerated pulp tissue.

Absence ofKu70Gene Obliterates X-Ray-InducedlacZMutagenesis of Small Deletions in Mouse Tissues

With the goal of understanding the role of non-homologous end-joining repair in the maintenance of genetic information at the tissue level, we studied mutations induced by radiation and subsequent repair of DNA double-strand breaks in Ku70 gene-deficient lacZ transgenic mice. The local mutation frequencies and types of mutations were analyzed on a lacZ gene that had been chromosomally integrated, which allowed us to monitor DNA sequence alterations within this 3.1-kbp region. The mutagenic process leading to the development of the most frequently observed small deletions in wild-type mice after exposure to 20 Gy of X rays was suppressed in Ku70(-/-) mice in the three tissues examined: spleen, liver and brain. Examination of DNA break rejoining and the phosphorylation of histone H2AX in Ku70-deficient and -proficient mice revealed that Ku70 deficiency decreased the frequency of DNA rejoining, suggesting that DNA rejoining is one of the causes of radiation-induced deletion mutations. Limited but statistically significant DNA rejoining was found in the liver and brain of Ku70-deficient mice 3.5 days after irradiation, showing the presence of a DNA double-strand break repair system other than non-homologous end joining. These data indicate a predominant role of non-homologous end joining in the production of radiation-induced mutations in vivo.

Analysis of the Runx2 promoter in osseous and non-osseous cells and identification of HIF2A as a potent transcription activator

Little is known about the upstream regulator of Runx2, a master regulator of osteoblast differentiation in bone tissues. To elucidate the molecular mechanism of Runx2 gene expression, we analyzed Runx2 promoter activity in osseous (MC3T3-E1, KS483, Kusa) and non-osseous (NIH3T3, C3H10T1/2, mouse embryonic fibroblasts) cells and also identified Runx2 upstream regulator using a Runx2 promoter-derived luciferase reporter system. After cloning 15 serial deletion constructs from − 6832 bp/+ 390 bp to − 37 bp/+ 390 bp of the Runx2-P1 promoter, we performed a transient transfection assay in osseous and non-osseous cells. A reduction in Runx2 promoter activity was observed in two regions; one was between − 3 kb and − 1 kb, and the other was between − 155 bp and − 75 bp. The step-down pattern in promoter activity between − 3 kb and − 1 kb was observed only in osseous cells. Interestingly, the step-down pattern between − 155 bp and − 75 bp was revealed in both cell types. Consistently, β-galactosidase staining in axial skeleton of − 3 kb-Runx2-P1- LacZ transgenic mice was positive, but that of all skeletal tissues of − 1 kb-Runx2-P1- LacZ transgenic mice was negative. To identify upstream regulators of the Runx2-P1 promoter, we screened 100 transcription factors using Runx2-P1-luciferase reporter constructs in NIH3T3 fibroblasts and HeLa cells. Among them, HIF2A was identified as the strongest activator of Runx2-P1 promoter activity. A HIF2A-responsive site on the Runx2 promoter was identified between − 106 bp and − 104 bp by mutation analysis. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed the binding of HIF2A to the Runx2-P1 promoter in vitro and in vivo, respectively. Knock-down using siRNA against HIF2A confirmed that HIF2A is an important regulator of Runx2 gene expression. Collectively, these results suggest that the region between − 3 kb and − 1 kb is required for the minimal skeletal tissue-specific expression of Runx2, and that the region between − 155 bp and − 75 bp is important for its basal transcription, which may be in part mediated by HIF2A in bone tissues.

A Map of Relationships Between Uterine Natural Killer Cells and Progesterone Receptor Expressing Cells During Mouse Pregnancy

Uterine natural killer (uNK) cells appear in implantation sites with decidualization. Initially, they are rare, highly proliferative cells that become abundant by midgestation and then die abruptly. Steps regulating the cyclic appearance of uNK cells are incompletely defined. Although progesterone (P4) has an essential role, mature uNK cells of women and mice lack progesterone receptors (PR). Immunohistochemical studies suggest that mouse PR − uNK cells may co-localize with PR + stromal cells while human PR − uNK cells co-localize with immature, DC-SIGN + dendritic cells (DCs). DCs have the potential to produce progesterone-regulated interleukin (IL)-15, a growth factor essential for uNK cells. Since the high affinity IL-15Rα is presented to differentiating NK cells in trans, requiring cell contact, histologically-detected interactions may be of central importance for uNK cell differentiation. Thus, a pregnancy time course, histological study of uNK cell differentiation and localization was undertaken in PR lacZ transgenic mice. PR + cells and uNK cells were co-localized using LacZ histochemistry and Dolichos biflorus (DBA) lectin staining, respectively. uNK cells appeared mesometrially, where PR + cells were rare, at gestation day 5.5. uNK cells had limited, apparently random contact with PR + cells throughout pregnancy and never themselves expressed PR. Thus, uNK cell differentiation does not appear to require contact with PR + cells.