Sequencing and characterization of the FVB/NJ mouse genome

Background

The FVB/NJ mouse strain has its origins in a colony of outbred Swiss mice established in 1935 at the National Institutes of Health. Mice derived from this source were selectively bred for sensitivity to histamine diphosphate and the B strain of Friend leukemia virus. This led to the establishment of the FVB/N inbred strain, which was subsequently imported to the Jackson Laboratory and designated FVB/NJ. The FVB/NJ mouse has several distinct characteristics, such as large pronuclear morphology, vigorous reproductive performance, and consistently large litters that make it highly desirable for transgenic strain production and general purpose use.

Results

Using next-generation sequencing technology, we have sequenced the genome of FVB/NJ to approximately 50-fold coverage, and have generated a comprehensive catalog of single nucleotide polymorphisms, small insertion/deletion polymorphisms, and structural variants, relative to the reference C57BL/6J genome. We have examined a previously identified quantitative trait locus for atherosclerosis susceptibility on chromosome 10 and identify several previously unknown candidate causal variants.

Conclusion

The sequencing of the FVB/NJ genome and generation of this catalog has increased the number of known variant sites in FVB/NJ by a factor of four, and will help accelerate the identification of the precise molecular variants that are responsible for phenotypes observed in this widely used strain.     

Sequencing and characterisation of the FVB/NJ mouse genome

ABSTRACT: BACKGROUND: The FVB/NJ mouse strain has its origins in a colony of outbred Swiss mice established in 1935 at the National Institutes of Health. Mice derived from this source were selectively bred for sensitivity to histamine diphosphate and the B strain of Friend leukemia virus. This led to the establishment of the FVB/N inbred strain, which was subsequently imported to the Jackson Laboratory and designated FVB/NJ. The FVB/NJ mouse has several distinct characteristics, such as large pronuclear morphology, vigorous reproductive performance, and consistently large litters that make it highly desirable for transgenic strain production and general purpose use. RESULTS: Using next-generation sequencing technology, we have sequenced the genome of FVB/NJ to approximately 50-fold coverage, and have generated a comprehensive catalog of single nucleotide polymorphisms, small insertion/deletion polymorphisms, and structural variants, relative to the reference C57BL/6J genome. We have examined a previously identified quantitative trait locus for atherosclerosis susceptibility on chromosome 10 and identify several previously unknown candidate causal variants. CONCLUSION: The sequencing of the FVB/NJ genome and generation of this catalog has increased the number of known variant sites in FVB/NJ by a factor of four, and will help accelerate the identification of the precise molecular variants that are responsible for phenotypes observed in this widely-used strain.     

Proteinuria of nonautoimmune origin in wild-type FVB/NJ mice

FVB/NJ mice frequently are used as transgenic hosts, but the suitability of this genetic background for transgenic and congenic models of systemic autoimmunity have not been reported. In this study, FVB/NJ mice were evaluated for the presence of serum autoantibodies and autoimmune kidney pathology. Previously unreported albuminuria was observed in aged female FVB/NJ mice; however, serum autoantibody testing, light microscopic evaluation of differentially stained renal sections, and evaluation of renal sections for immunoglobulin deposits revealed that the albuminuria was not of autoimmune etiology. Anecdotally, multiple characteristics of the FVB/NJ strain, including albuminuria, cholesterolemia, mild podocyte foot process effacement in aged female FVB/NJ kidneys and predisposition to enhanced Th2 immune responses, is reminiscent of human minimal change nephrotic syndrome (MCNS). We propose that mapping of genetic polymorphisms that are responsible for these traits in FVB/NJ mice may lead to increased understanding of mild nephrotic syndromes including MCNS and other proteinurias.     

Development of mammary hyperplasia and neoplasia in MMTV-TGF alpha transgenic mice

To study the role of transforming growth factor alpha (TGF alpha) in normal mammary development and mammary neoplasia in vivo, we have generated transgenic mice in which a human TGF alpha cDNA is expressed under the control of the MMTV enhancer/promoter. Overexpression of TGF alpha in the mammary epithelium, as confirmed by in situ hybridization and immunohistochemistry, is associated with hyperplasia of alveoli and terminal ducts in virgin female and pregnant transgenic mice. A range of morphologic abnormalities including lobular hyperplasia, cystic hyperplasia, adenoma, and adenocarcinoma is seen in mammary tissue of transgenic females. In contrast, no morphologic abnormalities are seen in transgenic males in spite of TGF alpha overexpression in salivary glands and reproductive organs. TGF alpha can therefore act as an oncogene in vivo and appears to predispose mammary epithelium to neoplasia and carcinoma.     

BALB/Mtv-null mice responding to strong mouse mammary tumor virus superantigens restrict mammary tumorigenesis

The absence of endogenous mouse mammary tumor viruses (MMTVs) in the congenic mouse strain, BALB/Mtv-null, restricts the early steps of exogenous C3H MMTV infection, preventing the superantigen (Sag) response and mammary tumorigenesis. Here we demonstrate that BALB/Mtv-null mice also resist tumor induction by FM MMTV, which encodes a stronger Sag compared to C3H MMTV. In contrast to infections with C3H MMTV, Mtv-null mice show FM-MMTV Sag-specific responses comparable to those observed in susceptible BALB/c mice. Neither virus shows significant replication in the spleen or mammary gland. Thus, Mtv-null mice restrict MMTV replication and mammary tumorigenesis even after a robust Sag response.     

Mapping hyper-susceptibility to colitis-associated colorectal cancer in FVB/NJ mice

Inbred strains of mice differ in susceptibility to colitis-associated colorectal cancer (CA-CRC). We tested 10 inbred strains of mice for their response to azoxymethane/dextran sulfate sodium-induced CA-CRC and identified a bimodal inter-strain distribution pattern when tumor multiplicity was used as a phenotypic marker of susceptibility. The FVB/NJ strain was particularly susceptible showing a higher tumor burden than any other susceptible strains (12.5-week post-treatment initiation). FVB/NJ hyper-susceptibility was detected as early as 8-week post-treatment initiation with FVB/NJ mice developing 5.5-fold more tumors than susceptible A/J or resistant B6 control mice. Linkage analysis by whole genome scan in informative (FVB/NJ×C3H/HeJ)F2 mice identified a novel susceptibility locus designated as C olon c ancer s usceptibility 6 (Ccs6) on proximal mouse chromosome 6. When gender was used as a covariate, a LOD score of 5.4 was computed with the peak marker being positioned at rs13478727, 43.8 Mbp. Mice homozygous for FVB/NJ alleles at this locus had increased tumor multiplicity compared to homozygous C3H/HeJ mice. Positional candidates in this region of chromosome 6 were analyzed with respect to a possible role in carcinogenesis and a role in inflammatory response using a new epigenetic gene scoring tool (Myeloid Inflammation Score).     

Effects of FVB/NJ and C57Bl/6J strain backgrounds on mammary tumor phenotype in inducible nitric oxide synthase deficient mice

The ability to genetically manipulate mice has led to rapid progress in our understanding of the roles of different gene products in human disease. Transgenic mice have often been created in the FVB/NJ (FVB) strain due to its high fecundity, while gene-targeted mice have been developed in the 129/SvJ-C57Bl/6J strains due to the capacity of 129/SvJ embryonic stem cells to facilitate germline transmission. Gene-targeted mice are commonly backcrossed into the C57Bl/6J (B6) background for comparison with existing data. Genetic modifiers have been shown to modulate mammary tumor latency in mouse models of breast cancer and it is commonly known that the FVB strain is susceptible to mammary tumors while the B6 strain is more resistant. Since gene-targeted mice in the B6 background are frequently bred into the polyomavirus middle T (PyMT) mouse model of breast cancer in the FVB strain, we have sought to understand the impact of the different genetic backgrounds on the resulting phenotype. We bred mice deficient in the inducible nitric oxide synthase (iNOS) until they were congenic in the PyMT model in the FVB and B6 strains. Our results reveal that the large difference in mean tumor latencies in the two backgrounds of 53 and 92 days respectively affect the ability to discern smaller differences in latency due to the Nos2 genetic mutation. Furthermore, the longer latency in the B6 strain enables a more detailed analysis of tumor formation indicating that individual tumor development is not stoichastic, but is initiated in the #1 glands and proceeds in early and late phases. NO production affects tumors that develop early suggesting an association of iNOS-induced NO with a more aggressive tumor phenotype, consistent with human clinical data positively correlating iNOS expression with breast cancer progression. An examination of lung metastases, which are significantly reduced in PyMT/iNOS^−/− mice compared with PyMT/iNOS^+/+ mice only in the B6 background, is concordant with these findings. Our data suggest that PyMT in the B6 background provides a useful model for the study of inflammation-induced breast cancer.     

Reorganization of horizontal cell processes in the developing FVB/N mouse retina

We investigated the morphological changes of horizontal cells after postnatal photoreceptor degeneration in the developing FVB/N mouse retina, using immunocytochemistry with anti-calbindin D-28K. From postnatal day 14 (P14) onwards, processes emerging from horizontal cells descend into the inner plexiform layer (IPL) and ramify mainly in stratum 1 of the IPL. Electron microscopy revealed that the descending processes make synaptic contacts with bipolar cells in the outer plexiform layer. Our results clearly demonstrate that loss of photoreceptor cells induces the reorganization of horizontal cell processes in the retinas of FVB/N mice as they mature.     

Estrogen receptor alpha is required for mammary development and the induction of mammary hyperplasia and epigenetic alterations in the aromatase transgenic mice

Aromatase transgenic mice exhibit hyperplastic and dysplastic changes, attesting to the importance of local estrogen in breast carcinogenesis. These mice also show increased levels of the estrogen receptor and β (ER, ERβ) suggesting that this receptor may play an important role in the initiation of estrogen-mediated mammary hyperplasia observed in these mice. To address the specific role of ER in the mammary development and in the induction of estrogen-mediated hyperplasia in aromatase transgenic mice, we have generated MMTV-aromatase × ER knockout cross (referred as aromatase/ERKO). Even though ERβ is expressed in aromatase/ERKO mice, lack of ER leads to impaired mammary growth in these mice. The data suggest that ER plays an important role in the mammary gland development as well as in the induction of mammary hyperplasia in aromatase transgenic mice. Lack of ER expression in the aromatase/ERKO mice resulted in a decrease in the expression of Cyclin D1, PCNA and TGFβ relative to the aromatase parental strain. The studies involving aromatase/ERKO mice show that lack of ER results in impaired mammary development even in the presence of continuous tissue estrogen, suggesting estrogen/ER-mediated actions are critical for mammary development and carcinogenesis.     

Early multipotential pituitary focal hyperplasia in the alpha-subunit of glycoprotein hormone-driven pituitary tumor-transforming gene transgenic mice

Pituitary tumor-transforming gene (PTTG), a securin protein isolated from pituitary tumor cell lines, is highly expressed in invasive tumors and exhibits characteristics of a transforming gene. To determine the role of PTTG in pituitary tumorigenesis, transgenic human PTTG1 was targeted to the mouse pituitary using the alpha-subunit of glycoprotein hormone. Males showed plurihormonal focal pituitary transgene expression with LH-, TSH-, and, unexpectedly, also GH-cell focal hyperplasia and adenoma, associated with increased serum LH, GH, testosterone, and/or IGF-I levels. MRI revealed both pituitary and prostate enlargement at 9-12 months. Urinary obstruction caused by prostatic hyperplasia and seminal vesicle hyperplasia, with renal tract inflammation, resulted in death by 10 months in some animals. Pituitary PTTG expression results in plurihormonal hyperplasia and hormone-secreting microadenomas with profound peripheral growth-stimulatory effects on the prostate and urinary tract. These results provide evidence for early pituitary plasticity, whereby PTTG overexpression results in a phenotype switch in early pituitary stem cells and promotes differentiated polyhormonal cell focal expansion.     

Peroxisomal localization in the developing mouse cerebellum: implications for neuronal abnormalities related to deficiencies in peroxisomes

In subjects with Zellweger syndrome, the most severe phenotype of peroxisomal biogenesis disorder, brain abnormalities include cortical dysplasia, neuronal heterotopia, and dysmyelination. To clarify the relationship between the lack of peroxisomes and neuronal abnormalities, we investigated peroxisomal localization in the mouse cerebellum, using double immunofluorescent staining for peroxisomal proteins. On immunostaining for peroxisomal matrix protein, while there are few peroxisomes in Purkinje cells, many locate in astroglia, especially soma of Bergmann glia. Clusters of peroxisomes were seen on the inferior side of the Purkinje cell layer in mice on postnatal days 3-5, and with time there was a shift to the superior side. The peroxisomal punctate pattern was seen to be radial and co-localized with Bergmann glial fibers. In cultured cells from the mouse cerebellum, peroxisomes were few in Purkinje cells, whereas many were evident in glial fibrillary acidic protein-positive cells. On the other hand, on immunostaining for peroxisomal membrane protein Pex14p, many particles were seen in Purkinje cells during all developmental stages, which means Purkinje cells possessed empty peroxisomal structures similar to findings of fibroblasts from the Zellweger patients. As peroxisomes in glial cells may control the development of neurons, the neuron-glial interaction and mechanisms of developing central nervous systems deserve ongoing attention.     

C3H/HeN mammary tumor-bearing mice develop type-specific neutralizing antibodies and group-specific precipitating antibodies for the mouse mammary tumor virus.

The development of mouse mammary tumor virus (MMTV)-neutralizing antibodies in various strains of mice was measured by their ability to neutralize the focus-forming capacity of a Kirsten sarcoma virus (C3H MMTV) pseudotype containing the MMTV envelope glycoprotein gp52. C3H/HeN, but not GR/N and RIII, mammary tumor-bearing mice were found to develop neutralizing antibodies to this pseudotype. In addition, non-tumor-bearing C3H/HeN, GR/N, RIII, NIH Swiss, C57BL/6, and BALB/c mice and 13 feral mice were also negative for neutralizing antibodies. The neutralization was immunoglobulin G mediated, and the antibodies of C3H/HeN mammary tumor-bearing mice were type specific and capable of distinguishing C3H and GR/N MMTVs from RIII and C3H/HeNf MMTVs. Precipitating antibodies were detected in sera from RIII and GR/N tumor-bearing mice, GR/N non-tumor-bearing mice, and six of the feral mice, although these same sera did not neutralize the Kirsten sarcoma virus (C3H MMTV) pseudotype. The results of this study and of a previous study demonstrate that C3H/HeN mammary tumor-bearing mice develop three functionally distinct antibody populations: (i) group-specific virus-precipitating antibodies; (ii) type-specific virus-neutralizing antibodies; and (iii) type-specific cytotoxic antibodies.