TP53 is commonly altered in human cancer, and Tp53 reactivation suppresses tumours in vivo in mice (TP53 and Tp53 are also known as p53). This strategy has proven difficult to implement therapeutically, and here we examine an alternative strategy by manipulating the p53 family members, Tp63 and Tp73 (also known as p63 and p73, respectively). The acidic transactivation-domain-bearing (TA) isoforms of p63 and p73 structurally and functionally resemble p53, whereas the ?N isoforms (lacking the acidic transactivation domain) of p63 and p73 are frequently overexpressed in cancer and act primarily in a dominant-negative fashion against p53, TAp63 and TAp73 to inhibit their tumour-suppressive functions. The p53 family interacts extensively in cellular processes that promote tumour suppression, such as apoptosis and autophagy, thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53 pathway. Here we show that deletion of the ?N isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP, the gene that encodes amylin, a 37-amino-acid peptide co-secreted with insulin by the ? cells of the pancreas. We found that IAPP is causally involved in this tumour regression and that amylin functions through the calcitonin receptor (CalcR) and receptor activity modifying protein 3 (RAMP3) to inhibit glycolysis and induce reactive oxygen species and apoptosis. Pramlintide, a synthetic analogue of amylin that is currently used to treat type 1 and type 2 diabetes, caused rapid tumour regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers.
Tyrosinase is the rate-limiting enzyme for the production of melanin pigmentation. In the mouse and other animals, homozygous null mutations in the Tyrosinase gene (Tyr) result in the absence of pigmentation, i.e. albinism. Here we used the CRISPR/Cas9 system to generate mono- and bi-allelic null mutations in the Tyr locus by zygote injection of two single-guide and Cas9 RNAs. Injection into C57BL/6N wild-type embryos resulted in one completely albino founder carrying two different Tyr mutations. In addition, three pigmentation mosaics and fully pigmented littermates were obtained that transmitted new mutant Tyr alleles to progeny in test crosses with albinos. Injection into Tyr heterozygous (B6CBAF1/J×FVB/NJ) zygotes resulted in the generation of numerous albinos and also mice with a graded range of albino mosaicism. Deep sequencing revealed that the majority of the albinos and the mosaics had more than two new mutant alleles. These visual phenotypes and molecular genotypes highlight the somatic mosaicism and allele complexity in founders that occurs for targeted genes during CRISPR/Cas9-mediated mutagenesis by zygote injection in mice.
The roles of microRNAs (miRNAs) and the miRNA processing machinery in the regulation of stem cell biology are not well understood. Here, we show that the p53 family member and p63 isoform, ?Np63, is a transcriptional activator of a cofactor critical for miRNA processing (DGCR8). This regulation gives rise to a unique miRNA signature resulting in reprogramming cells to multipotency. Strikingly, ?Np63(-/-) epidermal cells display profound defects in terminal differentiation and express a subset of markers and miRNAs present in embryonic stem cells and fibroblasts induced to pluripotency using Yamanaka factors. Moreover, ?Np63(-/-) epidermal cells transduced with an inducible DGCR8 plasmid can differentiate into multiple cell fates in vitro and in vivo. We found that human primary keratinocytes depleted of ?Np63 or DGCR8 can be reprogrammed in 6 d and express a unique miRNA and gene expression signature that is similar but not identical to human induced pluripotent stem cells. Our data reveal a role for ?Np63 in the transcriptional regulation of DGCR8 to reprogram adult somatic cells into multipotent stem cells.
We examined different weight ranges and hormone dosages to determine superovulation protocols for 6 mouse strains commonly used in genetic engineering: C57BL/6NHsd, B6(Cg)-Tyr(c-2J)/J, B6D2F1/Hsd, FVB/NHsd, BALB/cAnNCr, and Crl:CD1(ICR). Mice from each strain were divided into groups based on weight roughly corresponding to those of 3-, 4-, 5-, and 6-wk-old mice. Mice were treated with 5 IU pregnant mare serum gonadotropin (PMSG) and 5 IU human chorionic gonadotropin (HCG). The weights of mice that produced maximal numbers of oocytes in response to these doses were 14.2 g or less for C57BL/6NHsd, 13.7 g or less for B6(Cg)-Tyr(c-2J)/J, 6.0 to 9.9 g for B6D2F1/Hsd, 14.5 to 16.4 g for FVB/NHsd, 14.8 g or less for BALB/cAnNCr, and 23.5 g or more for Crl:CD1(ICR). We then compared PMSG dosages of 5 and 2.5 IU per mouse and determined whether 2 doses of PMSG (5 or 2.5 IU, depending on prior results) administered 1 wk apart, followed by the standard HCG injection, would produce more oocytes when compared to a single dose of PMSG. FVB, B6D2F1, BALB/c, and CD1 mice responded best to a single dose of 5 IU of each hormone, whereas B6(Cg)-Tyr (c-2J)/J mice produced more oocytes after 2.5 IU PMSG. Although C57BL/6 mice given the standard dose produced good numbers of oocytes, the number was higher after 2 doses of PMSG at 5 IU per dose. We conclude that response to superovulation can be optimized based on mouse strain, weight, and the dose and timing of hormone injection.
WWOX, the gene that spans the second most common human chromosomal fragile site, FRA16D, is inactivated in multiple human cancers and behaves as a suppressor of tumor growth. Since we are interested in understanding WWOX function in both normal and cancer tissues we generated mice harboring a conditional Wwox allele by flanking Exon 1 of the Wwox gene with LoxP sites. Wwox knockout (KO) mice were developed by breeding with transgenic mice carrying the Cre-recombinase gene under the control of the adenovirus EIIA promoter. We found that Wwox KO mice suffered from severe metabolic defect(s) resulting in growth retardation and all mice died by 3 wk of age. All Wwox KO mice displayed significant hypocapnia suggesting a state of metabolic acidosis. This finding and the known high expression of Wwox in kidney tubules suggest a role for Wwox in acid/base balance. Importantly, Wwox KO mice displayed histopathological and hematological signs of impaired hematopoiesis, leukopenia, and splenic atrophy. Impaired hematopoiesis can also be a contributing factor to metabolic acidosis and death. Hypoglycemia and hypocalcemia was also observed affecting the KO mice. In addition, bone metabolic defects were evident in Wwox KO mice. Bones were smaller and thinner having reduced bone volume as a consequence of a defect in mineralization. No evidence of spontaneous neoplasia was observed in Wwox KO mice. We have generated a new mouse model to inactivate the Wwox tumor suppressor gene conditionally. This will greatly facilitate the functional analysis of Wwox in adult mice and will allow investigating neoplastic transformation in specific target tissues.
Computer-based training is potentially a useful means of gaining proficiency in various aspects of laboratory animal science. The authors present an overview of the AALAS Learning Library (ALL), an internet-based training system that was established in 2003 and is widely used for technician certification training and for IACUC-mandated training. To evaluate the effectiveness of the ALL as a tool for general training and for achieving certification, the AALAS Online Learning Committee initiated a review of the online courses. The authors analyzed the numbers of users who accessed different types of courses and completed exams in those courses. They also correlated ALL usage with pass rates in technician certification exams. Results suggest that the ALL is a highly effective method of training, particularly for technician certification.
Since the late 1980s, the tools to generate mice with deletions of tumor suppressors have made it possible to study such deletions in the context of a whole animal. Deletion of some tumor suppressors results in viable mice while deletion of others yield embryo lethal phenotypes cementing the concept that genes that often go awry in cancer are also of developmental importance. More sophisticated mouse models were subsequently developed to delete a gene in a specific cell type at a specific time point. Additionally, incorporation of point mutations in a specific gene as observed in human tumors has also revealed their contributions to tumorigenesis. On the other hand, some models never develop cancer unless combined with other deletions suggesting a modifying role in tumorigenesis. This review will describe the technical aspects of generating these mice and provide examples of the outcomes obtained from alterations of different tumor suppressors.
Expression of 15-lipoxygenase-1 (15-LOX-1) is decreased in many human cancers; however, the mechanistic significance of its decreased expression has been difficult to determine because its mouse homolog 12/15-LOX has opposing functions. We generated a mouse model in which expression of a human 15-LOX-1 transgene was targeted to the intestinal epithelium via the villin promoter. Targeted expression was confirmed by real-time reverse transcription-polymerase chain reaction and immunoblotting. When the 15-LOX-1 transgene was expressed in colonic epithelial cells of two independent mouse lines (B6 and FVB), azoxymethane-inducible colonic tumorigenesis was suppressed (mean number of tumors: wild type [WT] = 8.2, 15-LOX-1(+/-) = 4.91, 15-LOX-1(+/+) = 3.57; WT vs 15-LOX-1(+/-) two-sided P = .003, WT vs 15-LOX-1(+/+) two-sided P < .001; n = 10-14 mice per group). 15-LOX-1 transgene expression was always decreased in the tumors that did develop. In the presence of expression of the 15-LOX-1 transgene, expression of tumor necrosis factor alpha and its target inducible nitric oxide synthase were decreased and activation of nuclear factor-kappa B in colonic epithelial cells was inhibited.
Strains of mice vary in their susceptibility to ultra-violet (UV) radiation-induced skin tumors. Some strains of hairless mice (homozygous for the spontaneous Hr(hr) mutation) are particularly susceptible to these tumors. The skin tumors that develop in hairless mice resemble, both at the morphologic and molecular levels, UV-induced squamous cell carcinomas (SCC) and their precursors in human. The most commonly employed hairless mice belong to the SKH1 stock. However, these mice are outbred and their genetic background is not characterized, which makes them a poor model for genetic studies. We have developed a new inbred strain from outbred SKH1 mice that we named SKHIN/Sprd (now at generation F31). In order to characterize the genetic background of this new strain, we genotyped a cohort of mice at F30 with 92 microsatellites and 140 single nucleotide polymorphisms (SNP) evenly distributed throughout the mouse genome. We also exposed SKHIN/Sprd mice to chronic UV irradiation and showed that they are as susceptible to UV-induced skin carcinogenesis as outbred SKH1 mice. In addition, we proved that, albeit with low efficiency, inbred SKHIN/Sprd mice are suitable for transgenic production by classical pronuclear microinjection. This new inbred strain will be useful for the development of transgenic and congenic strains on a hairless inbred background as well as the establishment of syngeneic tumor cell lines. These new tools can potentially help elucidate a number of features of the cutaneous response to UV irradiation in humans, including the effect of genetic background and modifier genes.
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