Sarah G. Shinpock

Genetic analysis in the Collaborative Cross breeding population

Genetic reference populations in model organisms are critical resources for systems genetic analysis of disease related phenotypes. The breeding history of these inbred panels may influence detectable allelic and phenotypic diversity. The existing panel of common inbred strains reflects historical selection biases, and existing recombinant inbred panels have low allelic diversity. All such populations may be subject to consequences of inbreeding depression. The Collaborative Cross (CC) is a mouse reference population with high allelic diversity that is being constructed using a randomized breeding design that systematically outcrosses eight founder strains, followed by inbreeding to obtain new recombinant inbred strains. Five of the eight founders are common laboratory strains, and three are wild-derived. Since its inception, the partially inbred CC has been characterized for physiological, morphological, and behavioral traits. The construction of this population provided a unique opportunity to observe phenotypic variation as new allelic combinations arose through intercrossing and inbreeding to create new stable genetic combinations. Processes including inbreeding depression and its impact on allelic and phenotypic diversity were assessed. Phenotypic variation in the CC breeding population exceeds that of existing mouse genetic reference populations due to both high founder genetic diversity and novel epistatic combinations. However, some focal evidence of allele purging was detected including a suggestive QTL for litter size in a location of changing allele frequency. Despite these inescapable pressures, high diversity and precision for genetic mapping remain. These results demonstrate the potential of the CC population once completed and highlight implications for development of related populations.

Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice

BackgroundAnalysis of an allelic series of point mutations in a gene, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, is a valuable method for discovering the full scope of its biological function. Here we present an efficient gene-driven approach for identifying ENU-induced point mutations in any gene in C57BL/6J mice. The advantage of such an approach is that it allows one to select any gene of interest in the mouse genome and to go directly from DNA sequence to mutant mice.ResultsWe produced the Cryopreserved Mutant Mouse Bank (CMMB), which is an archive of DNA, cDNA, tissues, and sperm from 4,000 G1 male offspring of ENU-treated C57BL/6J males mated to untreated C57BL/6J females. Each mouse in the CMMB carries a large number of random heterozygous point mutations throughout the genome. High-throughput Temperature Gradient Capillary Electrophoresis (TGCE) was employed to perform a 32-Mbp sequence-driven screen for mutations in 38 PCR amplicons from 11 genes in DNA and/or cDNA from the CMMB mice. DNA sequence analysis of heteroduplex-forming amplicons identified by TGCE revealed 22 mutations in 10 genes for an overall mutation frequency of 1 in 1.45 Mbp. All 22 mutations are single base pair substitutions, and nine of them (41%) result in nonconservative amino acid substitutions. Intracytoplasmic sperm injection (ICSI) of cryopreserved spermatozoa into B6D2F1 or C57BL/6J ova was used to recover mutant mice for nine of the mutations to date.ConclusionsThe inbred C57BL/6J CMMB, together with TGCE mutation screening and ICSI for the recovery of mutant mice, represents a valuable gene-driven approach for the functional annotation of the mammalian genome and for the generation of mouse models of human genetic diseases. The ability of ENU to induce mutations that cause various types of changes in proteins will provide additional insights into the functions of mammalian proteins that may not be detectable by knockout mutations.

Influence of thymus cells on erythropoiesis of parental marrow in irradiated hybrid mice.

Summary Living parental thymus cells injected into heavily irradiated F1 hybrid recipients augmented erythropoiesis resulting from transplanted bone marrow from the same, but not the second, parental strain. The augmentation increased as parental thymus cell dose was increased. Thymus cells alone, regardless of origin, gave rise to no erythropoiesis. When recipient spleen weight, 8 days after marrow transplantation, was used as a criterion of marrow growth, the data for parental cell doses 4 × 107 and above paralleled those from 59Fe-uptake studies in the same mice. Parental thymus cells were effective even when administered 1 to 2 days before or after the marrow transplant.

Further studies on the relationship of the thymus to hemopoiesis.

Thymocytes of parental (P) type did not augment hemopoiesis in irradiated hybrid (F1) recipients of P marrow when (1) implanted i.p. in Millipore diffusion chambers as cell suspensions or as tissue slices; (2) disrupted by freezing and thawing or by sonification; or (3) used as saline extracts. These results suggest strongly that a humoral factor is not responsible for the augmenting effect. The augmentation of early marrow growth in P → F1 chimeras, produced by a standard number of P thymocytes, was dependent on age of thymus donor (within the limits of 1–13 weeks in this study). The effectiveness of thymoeytes from donors of different ages was related inversely to cell yield. The ability of thymocytes to augment marrow growth was found in transplantation situations other than the particular P → F1 poor growth cases previously studied so extensively; these include the allogenic A.SW → B6D2F1 and xenogenic Sprague-Dawley rat → B6D2F1 combinations. Ninety-day mortality data from several P → F1 and allogenic marrow plus thymocyte transplantation experiments showed that thymus of marrow donor type, either P or allogenic to F1 recipients, decreased survival of chimeras by accelerating graft-versus-host (GVH) disease. However, all long-term survivors of this constitution, although few in number, retained marrow grafts, whereas those that had been given either host-type or no thymocetes occasionally reverted to host-type red cells.

Expression of the Globin Genes and Hematopoiesis in Beta-Thalassemic Mice

Mice homozygous for a deletion of the beta-dmajor globin gene exhibit clinical symptoms of human beta-thalassemia and are good experimental animals for investigating the regulation of globin gene expression, perturbation of hematopoiesis, and potential methods for treating patients with beta-thalassemia. Homozygous beta-thalassemic mice have a microcytic anemia, their red blood cells display anisocytosis, poikilocytosis and a shortened life span, and iron overloading occurs in several tissues in response to increased erythropoiesis2,3. Mice heterozygous for the beta-thalassemia1,2 mutation are clinically normal.

ABILITY OF THYMIC LYMPHOCYTES TO ALTER CFU KINETICS IN RADIATION CHIMERAS

It is known that the poor colony‐forming ability of B6 bone marrow transplanted into B6D2F1 hybrids can be improved if B6 lymphocytes are given in addition. It was recently reported that the augmenting lymphocytes decrease the doubling time of differentiating hemopoietic cells. to determine whether thymus cells alter the self‐renewal of CFUs in this parent F1 combination, retransplan‐tation and 3H‐thymidine ‘suicide’ were employed as methods to determine the cell‐division rate. We have observed that in the presence of thymocytes, parental bone marrow cells are seeded more efficiently in the spleen, and the lag phase of the CFUs growth curve is shortened. However, thymic lymphocytes do not increase the slope of the exponential growth phase of CFUs.