The X-linked imprinted gene family Fthl17 shows predominantly female expression following the two-cell stage in mouse embryos

Differences between male and female mammals are initiated by embryonic differentiation of the gonad into either a testis or an ovary. However, this may not be the sole determinant. There are reports that embryonic sex differentiation might precede and be independent of gonadal differentiation, but there is little molecular biological evidence for this. To test for sex differences in early-stage embryos, we separated male and female blastocysts using newly developed non-invasive sexing methods for transgenic mice expressing green fluorescent protein and compared the gene-expression patterns. From this screening, we found that the Fthl17 (ferritin, heavy polypeptide-like 17) family of genes was predominantly expressed in female blastocysts. This comprises seven genes that cluster on the X chromosome. Expression analysis based on DNA polymorphisms revealed that these genes are imprinted and expressed from the paternal X chromosome as early as the two-cell stage. Thus, by the time zygotic genome activation starts there are already differences in gene expression between male and female mouse embryos. This discovery will be important for the study of early sex differentiation, as clearly these differences arise before gonadal differentiation.     

Generation of a novel germline stem cell line expressing a germline‐specific reporter in the mouse

Germline stem (GS) cells are stem cell lines derived from postnatal male germline cells. Remarkably, GS cells can form functional spermatozoa when transplanted into infertile host mouse testes, indicating that GS cells have spermatogonial stem cell (SSC) activity. As GS cells are the only type with SSC activity, they are most suitable for in vitro studies on male germ cell differentiation. However, GS cells can deviate from the germ cell state to become other types of cells, depending on culture conditions. Therefore, it is desirable to have a monitor system to ensure that GS cells are kept at the germ cell state in culture. Here, we established GS cell lines from neonatal testes of transgenic mice that express the fluorescent protein, Venus, whose gene expression is driven by the promoter of Mvh (mouse Vasa homolog), a gene highly specific to mammalian germ cells. This novel cell line has genuine GS cell properties equivalent to existing GS lines, including the ability to generate viable offspring. This Mvh–Venus GS cell line, to our knowledge, is the first one expressing a germ cell‐specific reporter. This valuable resource should provide new opportunities for studies on male germ cell differentiation. genesis 51:498–505. © 2013 Wiley Periodicals, Inc.     

Modelling infectious diseases with relapse: a case study of HSV-2

Background

Herpes Simplex Virus Type 2 (HSV-2) is one of the most common sexually transmitted diseases. Although there is still no licensed vaccine for HSV-2, a theoretical investigation of the potential effects of a vaccine is considered important and has recently been conducted by several researchers. Although compartmental mathematical models were considered for each special case in the previous studies, as yet there are few global stability results.

Results

In this paper, we formulate a multi-group SVIRI epidemic model for HSV-2, which enables us to consider the effects of vaccination, of waning vaccine immunity, and of infection relapse. Since the number of groups is arbitrary, our model can be applied to various structures such as risk, sex, and age group structures. For our model, we define the basic reproduction number ?₀ and prove that if ?₀≤1, then the disease-free equilibrium is globally asymptotically stable, whereas if ?₀>1, then the endemic equilibrium is so. Based on this global stability result, we estimate ?₀ for HSV-2 by applying our model to the risk group structure and using US data from 2001 to 2014. Through sensitivity analysis, we find that ?₀ is approximately in the range of 2-3. Moreover, using the estimated parameters, we discuss the optimal vaccination strategy for the eradication of HSV-2.

Conclusions

Through discussion of the optimal vaccination strategy, we come to the following conclusions. (1) Improving vaccine efficacy is more effective than increasing the number of vaccines. (2) Although the transmission risk in female individuals is higher than that in male individuals, distributing the available vaccines almost equally between female and male individuals is more effective than concentrating them within the female population.

     

Knock-in of integrin beta 1D affects primary but not secondary myogenesis in mice

Integrins are extracellular matrix receptors composed of alpha and beta subunits involved in cell adhesion, migration and signal transduction. The beta1 subunit has two isoforms, beta 1A ubiquitously expressed and beta 1D restricted to striated muscle. They are not functionally equivalent. Replacement of beta 1A by beta 1D (beta 1D knock-in) in the mouse leads to midgestation lethality on a 50% Ola/50% FVB background [Baudoin, C., Goumans, M. J., Mummery, C. and Sonnenberg, A. (1998). Genes Dev. 12, 1202-1216]. We crossed the beta 1D knock-in line into a less penetrant genetic background. This led to an attenuation of the midgestation lethality and revealed a second period of lethality around birth. Midgestation death was apparently not caused by failure in cell migration, but rather by abnormal placentation. The beta 1D knock-in embryos that survived midgestation developed until birth, but exhibited severely reduced skeletal muscle mass. Quantification of myotube numbers showed that substitution of beta 1A with beta 1D impairs primary myogenesis with no direct effect on secondary myogenesis. Furthermore, long-term primary myotube survival was affected in beta 1D knock-in embryos. Finally, overexpression of beta 1D in C2C12 cells impaired myotube formation while overexpression of beta 1A primarily affected myotube maturation. Together these results demonstrate for the first time distinct roles for beta1 integrins in primary versus secondary myogenesis and that the beta 1A and beta 1D variants are not functionally equivalent in this process.     

Fluorescence-Guided Surgery of Liver Metastasis in Orthotopic Nude-Mouse Models

We report here the development of fluorescence-guided surgery of liver metastasis. HT29 human colon cancer cells expressing green fluorescent protein (GFP) were initially injected in the spleen of nude mice. Three weeks later, established liver metastases were harvested and implanted on the left lobe of the liver in other nude mice in order to make an orthotopic liver metastasis model. Fourteen mice with a single liver metastasis were randomized into bright-light surgery (BLS) or fluorescence-guided surgery (FGS) groups. Seven mice were treated with BLS, seven were treated with FGS. Three weeks after implantation, the left lobe of the liver with a single metastasis was exposed through a median abdominal incision. BLS was performed under white light. FGS was performed using a hand-held portable fluorescence imaging system (Dino-Lite). Post-surgical residual tumor fluorescence was visualized with the OV100 Small Animal Imaging System. Residual tumor fluorescence after BLS was clearly visualized at high magnification with the OV100. In contrast, residual tumor fluorescence after FGS was not detected even at high magnification with the OV100. These results demonstrate the feasibility of FGS for liver metastasis.     

Tumor-Targeting Salmonella typhimurium A1-R in Combination with Trastuzumab Eradicates HER-2-Positive Cervical Cancer Cells in Patient-Derived Mouse Models

We have previously developed mouse models of HER-2-positive cervical cancer. Tumors in nude mice had histological structures similar to the original tumor and were stained by anti-HER-2 antibody in the same pattern as the patient’s cancer. We have also previously developed tumor-targeting Salmonella typhimurium A1-R and have demonstrated its efficacy against patient-derived tumor mouse models, both alone and in combination. In the current study, we determined the efficacy of S. typhimurium A1-R in combination with trastuzumab on a patient-cancer nude-mouse model of HER-2 positive cervical cancer. Mice were randomized to 5 groups and treated as follows: (1) no treatment; (2) carboplatinum (30 mg/kg, ip, weekly, 5 weeks); (3) trastuzumab (20 mg/kg, ip, weekly, 5 weeks); (4) S. typhimurium A1-R (5 × 10⁷ CFU/body, ip, weekly, 5 weeks); (5) S. typhimurium A1-R (5 × 10⁷ CFU/body, ip, weekly, 5 weeks) + trastuzumab (20 mg/kg, ip, weekly, 5 weeks). All regimens had significant efficacy compared to the untreated mice. The relative tumor volume of S. typhimurium A1-R + trastuzumab-treated mice was smaller compared to trastuzumab alone (p = 0.007) and S. typhimurium A1-R alone (p = 0.039). No significant body weight loss was found compared to the no treatment group except for carboplatinum-treated mice (p = 0.021). Upon histological examination, viable tumor cells were not detected, and replaced by stromal cells in the tumors treated with S. typhimurium A1-R + trastuzumab. The results of the present study suggest that S. typhimurium A1-R and trastuzumab in combination are highly effective against HER-2-expressing cervical cancer.     

Improvement of Germ Line Transmission by Targeting β-galactosidase to Nuclei in Transgenic Mice

The Escherichia coli lacZ gene has frequently been used as a reporter in cell lineage analysis, in determining the elements regulating spatial and temporal gene expression, and in enhancer/gene trap detection of developmentally regulated genes. However, it is uncertain whether lacZ expression affects eukaryotic cell growth and development. By using a gene trap, we previously isolated the promoter, Ayu1, which is active in ES cells and in several tissues including the gonads. We used this promoter and the nuclear location signal of the SV40 large T gene to locate β-galactosidase either in the cytoplasm or the nucleus. Transgenic lines containing β-galactosidase in the cytoplasm of a wide variety of cell types did not transmit the transgene to their offspring. In contrast, transgenic mice, containing β-galactosidase in the nucleus, did transmit the transgene successfully. Interestingly, lacZ expression in the brain was more restricted when β-galactosidase activity was detected in the cytoplasm. These data suggested that cytoplasmic β-galactosidase affects certain developmental processes or gametogenesis resulting in transmission distortion of the transgene, and that this effect can be reduced by targeting β-galactosidase to the nucleus. We also found that Ayu1-driven lacZ expression in the duodenum of adult transgenic mice was sexually dimorphic, being positive in females and negative in males.