The rat is a widely used model in biomedical research and is often the preferred rodent model in many areas of physiological and pathobiological research. Although many genetic tools are available for the rat, methods to produce gene-disrupted knockout rats are greatly needed. In this study, we developed protocols for creating N-ethyl-N-nitrosourea (ENU)-induced germline mutations in several rat strains. F1 preweanling pups from mutagenized Sprague Dawley (SD) male rats were then screened for functional mutations in Brca1 and Brca2 using a yeast gap-repair, ADE2-reporter truncation assay. We produced knockout rats for each of these two breast cancer suppressor genes.

Breast cancer risk is a polygenic trait. To identify breast cancer modifier alleles that have a high population frequency and low penetrance we used a comparative genomics approach. Quantitative trait loci (QTL) were initially identified by linkage analysis in a rat mammary carcinogenesis model followed by verification in congenic rats carrying the specific QTL allele under study. The breast cancer genetics ͉ cancer epidemiology ͉ comparative genomics ͉ noncoding elements ͉ rat models D espite immense efforts, the search for modifier genes underlying complex diseases has not been highly productive. Alleles of modifier genes that influence common disease risk have a moderate-to-high population frequency with a low penetrance. It has been suggested that alleles acting in this manner comprise the majority of genetic risk for many common diseases such as breast cancer (1, 2). It is estimated that if most risk alleles were identified, it would become possible to assign Ϸ90% of breast cancer risk to 50% of women (3). In most studies, candidate modifier genes are selected based on function, such as DNA repair or estrogen metabolism for breast cancer. Over 100 such candidate modifier genes have been tested in breast cancer case-control association studies (Ͼ400 SNPs); few show a consistent and significant association with risk in large sample populations (4). These results suggest the need for an alternative strategy to identify breast cancer modifier genes. Our laboratory has pursued the identification of candidate loci by using wholegenome linkage studies in inbred rat mammary cancer models, followed by fine-mapping in congenic rats. The rat was chosen because, similar to humans, it develops mammary carcinomas that are hormone-responsive and of ductal origin (5, 6). Using a backcross of [Wistar-Kyoto (WKy) ϫ Wistar-Furth (WF)]F 1 ϫ WF rats, we identified four mammary carcinoma susceptibility QTL, Mcs5, Mcs6, Mcs7, and Mcs8, on rat chromosomes 5, 7, 10, and 14, respectively (7). The WKy allele of Mcs5 acts to suppress mammary tumor multiplicity in a susceptible WF genetic background and has been shown to consist of at least three clustered loci; among these, Mcs5a confers a phenotype of resistance to mammary cancer (8). Here we show that Mcs5a is a compound QTL located in a noncoding genomic region. We identified polymorphisms within the human genomic region orthologous to rat Mcs5a that significantly associate with breast cancer risk in women. ResultsTo further analyze the Mcs5a locus, WF.WKy congenic rat lines with different segments of the WKy allele were established and phenotyped for resistance to 7,12-dimethylbenzanthracene (DMBA)-induced carcinogenesis (Fig. 1). Mammary carcinoma susceptibility in DMBA-treated rats was reduced Ϸ50% for each congenic line O, WW, and XX (Fig. 1). The boundaries of the Mcs5a locus are given by the overlapping WKy sequences of congenic lines WW and XX, which define a genomic interval of Ϸ116 kb containing Mcs5a. By incorporating phenotypic data from additional congenic lines within ...

Evidence exists that BRCA2 carriers may have an elevated risk of breast, ovarian, colon, prostate, and pancreatic cancer. In general, carriers are defined as individuals with protein truncating mutations within the BRCA2 gene. Many Brca2 knockout lines have been produced and characterized in the mouse. We previously produced a rat Brca2 knockout strain in which there is a nonsense mutation in exon 11 between BRC repeats 2 and 3, and a truncated protein is produced. Interestingly, while such a mutation in homozygous mice would lead to limited survival of approximately 3 months, the Brca2 À/À rats are 100% viable and the vast majority live to over 1 year of age. Brca2 À/À rats show a phenotype of growth inhibition and sterility in both sexes. Aspermatogenesis in the Brca2 À/À rats is due to a failure of homologous chromosome synapsis. Long-term phenotypes include underdeveloped mammary glands, cataract formation and lifespan shortening due to the development of tumors and cancers in multiple organs. The establishment of the rat Brca2 knockout model provides a means to study the role of Brca2 in increasing cancer susceptibility and inducing a novel ocular phenotype not previously associated with this gene.

Introduction: Asparagus (Asparagus officinalis) is a perennial flowering plant species. Its main components have tumor-prevention, immune system-enhancement, and anti-inflammation effects. Network pharmacology is a powerful approach that is being applied increasingly to research of herbal medicines. Herb identification, study of compound targets, network construction, and network analysis have been used to elucidate how herbal medicines work. However, the interaction of bioactive substances from asparagus with the targets involved in multiple myeloma (MM) has not been elucidated. We explored the mechanism of action of asparagus in MM through network pharmacology and experimental verification.Methods: The active ingredients and corresponding targets of asparagus were acquired from the Traditional Chinese Medicine System Pharmacology database, followed by identification of MM-related target genes using GeneCards and Online Mendelian Inheritance in Man databases, which were matched with the potential targets of asparagus. Potential targets were identified and a target network of traditional Chinese medicine was constructed. The STRING database and Cytoscape were utilized to create protein–protein interaction (PPI) networks and further screening of core targets.Results: The intersection of target genes and core target genes of the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway was enriched, the top-five core target genes were selected, and the binding affinity of corresponding compounds to the top-five core targets was analyzed using molecular docking. Network pharmacology identified nine active components of asparagus from databases based on oral bioavailability and drug similarity, and predicted 157 potential targets related to asparagus. Enrichment analyses showed that “steroid receptor activity” and the “PI3K/AKT signaling pathway” were the most enriched biological process and signaling pathway, respectively. According to the top-10 core genes and targets of the PPI pathway, AKT1, interleukin (IL)-6, vascular endothelial growth factor (VEGF)A, MYC, and epidermal growth factor receptor (EGFR) were selected for molecular docking. The latter showed that five core targets of the PI3K/AKT signaling pathway could bind to quercetin, among which EGFR, IL-6, and MYC showed strong docking, and the diosgenin ligand could bind to VEGFA. Cell experiments showed that asparagus, through the PI3K/AKT/NF-κB pathway, inhibited the proliferation and migration of MM cells, and caused retardation and apoptosis of MM cells in the G0/G1 phase.Discussion: In this study, the anti-cancer activity of asparagus against MM was demonstrated using network pharmacology, and potential pharmacological mechanisms were inferred using in vitro experimental data.