Carol C. Linder

Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice

Targeted mutagenesis in mice, a powerful tool for the analysis of gene function and human disease, makes extensive use of 129 mouse substrains. Although all are named 129, we document that outcrossing of these substrains, both deliberate and accidental, has lead to extensive genetic variability among substrains and embryonic stem cells derived from them. This clearer understanding of 129 substrain variability allows consideration of its negative impact on targeting technology, including: homologous recombination frequencies, preparation of inbred animals, and availability of appropriate controls. Based on these considerations we suggest a number of recommendations for future experimental design.

CFTR expression is regulated during both the cycle of the seminiferous epithelium and the oestrous cycle of rodents.

Severely reduced fertility is a common finding in cystic fibrosis (CF). We used in situ hybridization to examine the cell–specific expression of CFTR in the reproductive organs of rodents. In males CFTR mRNA is found in the round spermatids (spermatogenic stages V–X) and in the principal cells that line the initial segment of the epididymis. In both the testis and the epididymis, CFTR expression is developmentally regulated suggesting that the defect in the genital tract of male CF patients is of developmental origin. CFTR expression in the luminal and glandular epithelium of the uterus is regulated during the oestrous cycle and is maximal at pro–oestrus. Our results provide a biological rationale for the reduced fertility of CF patients, and suggest a possible cause for the comparatively poorer prognosis for women with CF.

The molecular biology of the FSH receptor

The actions of follicle stimulating hormone (FSH) mediated through its receptor are necessary for the proper functioning of mammalian gonads. The FSH receptor is localized on granulosa cells of the ovary and Sertoli cells of the testis. The expression of the FSH receptor (FSHR) in Sertoli cells varies in vivo as a function of the stage of the cycle of the seminiferous epithelium and in culture as a result of the addition of exogenous hormones. The gene for the FSH receptor is large and has been shown to be related in structure to the genes for luteinizing hormone (LH) receptor and thyroid stimulating hormone (TSH) receptor. The promoter region of the gene for FSHR does not contain a TATA box and has multiple transcriptional start sites. Less than 280 bp of the promoter are sufficient in transient transfection assays to direct expression of the chloramphenicol acetyl transferase gene (CAT) in a number of different cell types including non-gonadal cells. However, the promoter does direct the expression of a marker gene only into testis and ovary of transgenic mice.

Expression of Receptors during the Cycle of the Seminiferous Epitheliuma

The data presented in this manuscript are all based on some inferences drawn from past experimental observations. The first is that the synchronized testis model is representative of the normal testis. Support for this premise comes from the studies on SGP-2, SGP-1 and transferrin where results using in situ hybridization and Northern blots are similar for normal and for synchronized testes. The second inference is that normalizing all of the data to the relative levels of SGP-1 mRNA adjusts for the possible differential loading of mRNA samples. The logic of this practice is based on the observation obtained using in situ hybridization that SGP-1 mRNA levels did not change across the cycle. The third assumption was that total mRNA levels do not change greatly across the cycle. Wholesale changes in testicular mRNA such as the doubling of all of the mRNA transcripts per testis would not be accounted for by these studies. We feel that this is an unlikely complication because of the strong correlation between much of the data and the known biology. In addition, there is a strong correlation between our data on the FSHR mRNA and the binding data for FSH obtained by another laboratory and different techniques. The available data in the literature reveals that most of the Sertoli cell products which change in relative concentrations during the cycle of the seminiferous epithelium appear to have a maximum in either stages VII or IX or in stages XIII to III. Thus, the Sertoli cells in the cycle could be described has having two different functional modes. In mode A maximal levels of mRNA for a specific Sertoli cell product are roughly found in stages VII-IX and in mode B the maximal levels are found in stages XIII to III (Fig. 5). The distribution of the receptor mRNA and ABP mRNA can also be described in terms of these two modes of Sertoli cell function. Transferrin receptors, retinoic acid receptors, and androgen-binding protein appear to fall into mode A while FSH and androgen receptors fall into mode B (Table 1). Products which have antithetical functions such as FSH and inhibin or cystatin and cathepsin L are found in different modes. We propose that most of the actions of the Sertoli cell during the cycle can be specified by the dual modes described above rather than by an infinite number of operational modes.(ABSTRACT TRUNCATED AT 400 WORDS)

Mouse mutations as models for studying cataracts.

Human cataracts may be congenital or appear later in life; nuclear, cortical and lens epithelial proliferation forms have all been described. Due to the high degree of genetic homology, it is not surprising that multiple spontaneous or induced single gene mutations result in morphologically similar forms in the mouse. There are many different mutations in inbred strains of mice in which cataracts are a part of the phenotype. These mutations are of potential value in deciphering the molecular mechanisms involved in cataract formation. This report reviews the currently published cataract mutations in mice.

Strains, stocks and mutant mice.

The aim of this chapter is to provide the reader with an introduction to the various types of mouse strains and stocks commonly used in research today. This chapter outlines how these mice were generally created and maintained, describes their primary use in research and briefly discusses the advantages and shortcomings of each. In addition, the standardized nomenclature for each set of stocks and strains is briefly discussed. The complete rules set forth by the International Committee on Standardized Genetic Nomenclature for Mice are available on the internet through Mouse Genome Informatics (MGI) ( http://www.informatics.jax.org ). This chapter also summarizes information on commonly used resources that are exceptionally handy when working in mouse genetics, including databases with biological data, handbooks for husbandry and information on how to find and obtain the mice needed for your research.

Breeding systems: considerations, genetic fundamentals, genetic background, and strain types.

Publisher Summary The use of mice in biomedical research greatly facilitates the understanding of human biology and is critical for the development of treatment for diseases. The phenotypic characteristics and pathophysiology of mice carrying spontaneous or genetically engineered mutations are often attributed solely to alterations in the mutant gene. Inbred strains, outbred stocks, mice carrying genetically engineered and spontaneous mutations, congenic strains, recombinant inbred panels, and advanced intercross lines all represent a bounty of diverse strain types, distinct in their usefulness for numerous studies and currently available to researchers. These strains permit the study of not only single gene traits in both the normal and diseased context but also polygenic, complex traits and gene cluster regulation. Although the tools for genetic analysis have advanced by an astonishing degree in the past few decades, the fundamental concerns surrounding strain development and colony maintenance remain the same and include viability, fecundity, the availability of appropriate controls, the degree of genetic diversity, and the complexities caused by modifier genes and segregating backgrounds. This chapter is intended to describe commonly used strain types, outline appropriate breeding systems and controls, and offer insights into the caveats and opportunities that come with these various strains and their breeding schemes. This chapter also provides an overview of genetic fundamentals and breeding strategies for almost all the different strain types that a person is likely to encounter.

Applying mouse genetics expertise to research.

Carol Linder, PhD, Associate Professor of Biology, Department of Biology and Chemistry, New Mexico Highlands University, Las Vegas, NM.

Expression of the FSH Receptor in the Sertoli Cells

The regulation of cell-specific genes could result from the interaction of unique transcription factors with specific promoter sequences. In the testes of mammals, follicle stimulating hormone receptors (FSH-Rs) are apparently located only on the Sertoli cells. Thus, the gene for the FSH-R and the gene for the mullerian inhibiting substance (MIS) are the only gene products known that are unique to the Sertoli cells. Our interest in gene expression in the Sertoli cells has stimulated our studies of the expression of the FSH-R gene in an effort to dissect important cell-specific regulatory elements.