Laurie A. Shepel

Production of knockout rats using ENU mutagenesis and a yeast-basedscreening assay

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.

Mapping of 55 new rat microsatellite markers from chromosome-specific libraries

Abstract. Fifty-five novel rat microsatellite markers were isolated from libraries specific for rat chromosomes (Chrs) 1, 2, and 7. The markers were mapped in three backcross rat populations. Thirty of these markers mapped to Chrs 1, 2, or 7, while the other 25 mapped to other chromosomes. New markers for two genes, liver-specific transporter gene (Livtr) and insulin-responsive glucose transporter (Glut4), were also mapped to rat Chrs 9 and 10, respectively. Three provisionally assigned markers from previous studies were also confirmed. Detailed methodologies for the generation and enrichment of clones containing repeat sequences and for the isolation of chromosome-specific markers are presented, since they represent unique combinations and modifications of previous protocols. Such methods and the newly presented markers should be useful for both specific and general mapping studies in the rat.

A comparative analysis of allelic imbalance events in chemically induced rat mammary, colon, and bladder tumors

In this paper, patterns of allelic imbalances (AIs) in chemically induced rat mammary, colon, and bladder tumors from (Wistar Furth × Fischer 344)F1 rats are described and compared. Male F1 rats were administered azoxymethane (AOM), and colon tumors were collected at 58 wk after treatment. Female F1 rats were given either N‐nitroso‐N‐methylurea (NMU) or N‐butyl‐(hydroxybutyl)‐nitrosoamine (BBN), and mammary and bladder tumors were collected at 15 and 52 wk after treatment, respectively. DNA was extracted from a subset of 18 of the largest tumors from each group, and a genome scan was performed by using polymerase chain reaction and 90 polymorphic microsatellite markers. AIs, such as loss of heterozygosity, gene duplication, and microsatellite instability, were observed at low frequencies in all of the tumor models. Thirty random AIs were observed in the AOM‐induced colon tumors but only four in the NMU‐induced mammary tumors. In both these models, all the tumors were classified as adenocarcinomas, and most of the AIs observed were confined to single tumors with atypical histopathology. In contrast, 27 random AIs were identified in the BBN‐induced bladder tumors. AIs were observed in both transitional‐cell carcinomas and papillomas, although most were in the carcinomas. Statistical analysis of the AI data revealed no significant nonrandom AIs within or among the tumor models, although several of the infrequently observed AI events identified in the rat tumors may also be observed in the corresponding human tumor type. Mol. Carcinog. 24:47–56, 1999.

LINKAGE MAPPING OF RAT CHROMOSOME 5 MARKERS GENERATED FROM CHROMOSOME-SPECIFIC LIBRARIES

Abstract. Seventy-six novel microsatellite markers with various simple sequence repeat (SSR) motifs are reported in this paper. They were generated on the basis of non-radioactive library screening procedures from flow-sorted rat Chromosome (Chr) 5-specific DNA, and were mapped in three rat backcross populations. Fifty-four of these markers mapped to Chr 5, while the other 22 mapped to other chromosomes of the rat genome. The marker D3Uwm8 is a new microsatellite marker for the rat syndecan 4 (ryudocan) gene. A genotyping protocol based on agarose gel electrophoresis is also provided in this paper.

Genetic linkage mapping of 11 novel DNA markers and the ceruloplasmin (Cp) gene on rat chromosome 2

We have mapped 11 novel, anonymous genetic markers to rat chromosome 2. The rat ceruloplasmin gene (Cp) had been previously mapped to chromosomes 2 and 7q11-->q13 by two different methods. To resolve the assignment and to localize the Cp gene on the rat genetic linkage map, we used linkage analysis to confirm that rat Cp lies on chromosome 2.

Genetic mapping of the rat Lcn2 gene to chromosome 3

The expression of rat 24p3, encoded by the Lcn2 gene, has been associated with rat mammary carcinomas initiated by the neu oncogene (Stoesz and Gould, 1995). In this study, we assign the Lcn2 gene to rat chromosome band 3q12 by genetic linkage analysis.