The ter mutation responsible for germ cell deficiency but not testicular nor ovarian teratocarcinogenesis in ter / ter congenic mice

The ter (teratoma) gene causes germ cell deficiency and a high incidence of congenital testicular teratomas derived from primordial germ cells in 129/Sv- ter strain mice. Ovarian teratomas in LTXBJ mice originate from ovarian parthenotes. In order to study the function of the ter gene in germ cell development and teratocarcinogenesis, we examined the influence of a foreign genetic background on the ter action by introducing the ter gene of 129/Sv- ter strain mice into C57BL/6J, LTXBJ and C3H/HeJ genetic backgrounds by the backcross method and by thus establishing B6- ter , LTXBJ- ter and C3H- ter ter congenic strains, respectively. Histological analysis showed that germ cell deficiency occurred in both sexes of the ter mutants, through the fetal stages to adulthood, but that congenital testicular teratocarcinogenesis did not occur after the fifth backcross generation. The ter/ter gonads were smaller than normal (+/+ or +/ ter ). Experimental testicular teratomas never developed from intratesticular grafts of B6- ter genital ridges. LTXBJ- ter/ter females had no ovarian teratomas. It is concluded that the ter gene is solely responsible for germ cell deficiency, but not testicular teratocarcinogenesis, in ter congenic strains having background genes other than 129/Sv- ter and that the ter gene is not involved in ovarian teratocarcinogenesis.     

Interpretation of knockout experiments: the congenic footprint

In gene targeting experiments, the importance of genetic background is now widely appreciated, and knockout alleles are routinely backcrossed onto a standard inbred background. This produces a congenic strain with a substantial segment of embryonic stem (ES)-cell-derived chromosome still flanking the knockout allele, a phenomenon often neglected in knockout studies. In cholecystokynin 2 (Cckbr) knockout mice backcrossed with C57BL/6, we have found a clear 'congenic footprint' of expression differences in at least 10 genes across 40 Mb sequence flanking the Cckbr locus, each of which is potentially responsible for aspects of the 'knockout' phenotype. The expression differences are overwhelmingly in the knockout-low direction, which may point to a general phenomenon of background dependence. This finding emphasizes the need for caution in using gene knockouts to attribute phenotypic effects to genes. This is especially the case when the gene is of unknown function or the phenotype is unexpected, and is a particular concern for large-scale knockout and phenotypic screening programmes. However, the impact of genetic background should not be simply viewed as a potential confound, but as a unique opportunity to study the broader responses of a system to a specific (genetic) perturbation.     

Developmental expression of insulin-like growth factor II receptor (IGF-IIR) in congenic mouse embryonic lungs: Correlation between IGF-IIR mRNA and protein levels and heterochronic lung development

Embryonic lung maturation in the H-2 congenic pair, B10.A and B10, proceeds at different rates. The dependence of this heterochronic development on maternal haplotype suggests the involvement of a parentally imprinted gene. Since B10.A (H-2^b) and B10 (H-2^b) mice are genetically identical except for a 3-18 cM region of chromosome 17 that includes the H-2 complex, we sought a promising candidate gene(s) involved in regulating the rate of lung development from genes encoded in this region. The best candidate is the gene encoding the type II insulin-like growth factor receptor (IGF-IIR), whose ligand is the growth factor IGF-II. Only the maternal copy of this gene is expressed in postimplantation embryos. This receptor does not appear to transduce mitogenic signals; instead, IGF-IIR appears to regulate the levels of its ligand available to the growth-promoting type I IGF receptor (IGF-IR). Using in situ hybridization and indirect immunofluorescence, we demonstrate that IGF-IIR mRNA and protein are localized throughout the pulmonary mesenchyme, as well as in branching epithelia of the pseudoglandular and canalicular stages. We also examined the levels of IGF-IIR mRNA and protein expression by RNase protection assay and ligand blotting during the embryonic period of lung development in B10.A and B10 mice, and found that there is a highly significant positive correlation of IGF IIR levels with progressive development in both strains. Further, slower developing B10.A lungs contain significantly higher levels of IGF-IIR mRNA and protein than the more rapidly developing B10 lungs. These results suggest that haplotype-dependent elevation of IGF-IIR levels reduces the available concentration of IGF-II, resulting in a decreased rate of morphogenesis in B10.A mice. Heterochronic lung maturation, then, appears consequent to variable extracellular levels of this important growth factor. These results may be of clinical importance to predicting susceptibility to Respiratory Distress Syndrome in prenatal newborns. © Wiley-Liss, Inc.     

TheN haplotype of the murineβ-glucuronidase gene is altered in both its systemic regulation and its response to androgen induction

A new haplotype of the -glucuronidase gene complex, [Gus]^N, has been characterized following its transfer from the PAC/Cr strain to the standard strain C57BL/6J. TheN haplotype contains a novel structural gene allele which encodes an allozyme differing from all previously characterized allozymes in both size and charge. Altered systemic regulation is exhibited by the [Gus]^N haplotype. Multiple tissues contain levels of GUS protein that are 60±15% those found in the standardB haplotype. The regulatory mechanism for reduction is complex, involving tissue-specific changes in both enzyme synthesis and enzyme turnover. The changes in GUS protein synthesis do not result from changes in GUS mRNA levels. Instead, the amount of mature enzyme formed per mRNA molecule, or translational yield, is altered. These regulatory changes parallel those seen in other systemic regulatory variants of GUS which are also altered in translational yield. A commonality of mechanism among systemic regulatory variants of this gene is suggested. TheN haplotype is also exceptional in the nature of its response to androgenic induction in kidney proximal tubule epithelial cells. The time course for GUS induction consists of a lag period followed by a progressive increase in mRNA, rate of enzyme synthesis, and enzyme activity. For the [Gus]^N haplotype the lag is of an exceptionally short duration and the plateau is of a greater magnitude than for any haplotype previously described.This work was supported by United States Health Service Research Grant GM 31656.     

Genetic dissection of quantitative trait locus for ethanol sensitivity in long- and short-sleep mice

Interval-specific congenic strains (ISCS) allow fine mapping of a quantitative trait locus (QTL), narrowing its confidence interval by an order of magnitude or more. In earlier work, we mapped four QTL specifying differential ethanol sensitivity, assessed by loss of righting reflex because of ethanol (LORE), in the inbred long-sleep (ILS) and inbred short-sleep (ISS) strains, accounting for approximately 50% of the genetic variance for this trait. Subsequently, we generated reciprocal congenic strains in which each full QTL interval from ILS was bred onto the ISS background and vice versa. An earlier paper reported construction and results of the ISCS on the ISS background; here, we describe this process and report results on the ILS background. We developed multiple ISCS for each Lore QTL in which the QTL interval was broken into a number of smaller intervals. For each of the four QTL regions (chromosomes 1, 2, 11 and 15), we were successful in reducing the intervals significantly. Multiple, positive strains were overlapped to generate a single, reduced interval. Subsequently, this reduced region was overlaid on previous reductions from the ISS background congenics, resulting in substantial reductions in all QTL regions by approximately 75% from the initial mapping study. Genes with sequence or expression polymorphisms in the reduced intervals are potential candidates; evidence for these is presented. Genetic background effects can be important in detection of single QTL; combining this information with the generation of congenics on both backgrounds, as described here, is a powerful approach for fine mapping QTL.     

Congenic C57BL/6 mu opiate receptor (MOR) knockout mice: baseline and opiate effects

Homozygous µ-opioid receptor (MOR) knockout (KO) mice developed on a chimeric C57B6/129SV background lack morphine-induced antinociception, locomotion and reward. Therefore it appears that MOR largely mediates these morphine actions. However, one factor that could affect the extent of knockout deficits in morphine-induced behavior is the genetic background against which the gene deletion is expressed. To examine the effect of genetic background chimeric C57B6/129SV MOR knockout mice from the 15th generation of those developed in our laboratory were backcrossed for 10 successive generations with C57BL/6 mice, a strain which is more sensitive to many of the properties of morphine, to produce congenic MOR (conMOR) KO mice. Heterozygote conMOR KO mice display attenuated morphine locomotion and reduced morphine analgesia compared to wild-type mice. Homozygote conMOR KO mice display baseline hyperalgesia, no morphine place preference, no morphine analgesia and no morphine locomotion. These results are not qualitatively different from those observed in the MOR KO strain with a chimeric C57B6/129SV background, and suggest that although the strain has separate influences on these functions, it does not substantially interact with deletion of the µ opiate receptor gene.     

The OSUMMER lines: A series of ultraviolet-accelerated NRAS-mutant mouse melanoma cell lines syngeneic to C57BL/6

An increasing number of cancer subtypes are treated with front-line immunotherapy. However, approaches to overcome primary and acquired resistance remain limited. Preclinical mouse models are often used to investigate resistance mechanisms, novel drug combinations, and delivery methods; yet most of these models lack the genetic diversity and mutational patterns observed in human tumors. Here we describe a series of 13 C57BL/6J melanoma cell lines to address this gap in the field. The Ohio State University-Moffitt Melanoma Exposed to Radiation (OSUMMER) cell lines are derived from mice expressing endogenous, melanocyte-specific, and clinically relevant Nras driver mutations (Q61R, Q61K, or Q61L). Exposure of these animals to a single, non-burning dose of ultraviolet B accelerates the onset of spontaneous melanomas with mutational patterns akin to human disease. Furthermore, in vivo irradiation selects against potent tumor antigens, which could prevent the outgrowth of syngeneic cell transfers. Each OSUMMER cell line possesses distinct in vitro growth properties, trametinib sensitivity, mutational signatures, and predicted antigenicity. Analysis of OSUMMER allografts shows a correlation between strong, predicted antigenicity and poor tumor outgrowth. These data suggest that the OSUMMER lines will be a valuable tool for modeling the heterogeneous responses of human melanomas to targeted and immune-based therapies.