CORE B The objective of the Cell Biology Core (Core B) is to enable goals of the Program by providing testes, ovaries and enriched germ cells from mice of specific ages to support ChlP-seq experiments in Projects B Petkov, C Hibbs and D Handel;RNA-seq experiments in Projects C Hibbs and D Handel;and RNA and protein expression analyses in Project D Handel. Core B will utilize carefully controlled methods to obtain germ cells from juvenile testes for assays that follow the leading edge of the highly synchronized first wave of spermatogenesis. Additionally, Core B will use a technique of cell sedimentation at unit gravity to enrich pachytene spermatocytes and round sperm fids from adult testes. The Core will perform initial steps of RNA purification and chromatin-immunoprecipitation for experiments proposed in the Projects. Core B will be under the direction of Dr. Handel, who is highly experienced in investigation of meiosis and male germ-cell biology, and has expertise in isolation of mouse spermatocytes. Taken together, the functions of Core B ensure that all Projects will have high-quality and consistent experimental materials to achieve the individual Project goals as well as to collectively achieve the overall goals of the Program. 155 Cell Biology Core (Handel) Paigen, Kenneth SPECIFIC AIMS The Cell Biology Core (Core B) will support cellular and molecular needs of the Program. This goal will ensure high-quality and consistent preparations of germ cells and germ-cell extracts across all Projects, so that experimental results in the Program will be directly comparable. Moreover, centralizing these functions into a Core, under the direction of an experienced leader, provides efficiencies of cost and function. The Core will provide material for experiments that utilize gonads or germ cells, including expression analyses (Project D Handel), RNA-seq (Projects C Hibbs and D Handel), and ChlP-seq (Projects B Petkov, C Hibbs and D Handel).
The aims for the Cell Biology Core are:
Specific Aim 1. The Core will retrieve gonads from mice and prepare enriched germ cells from juvenile and mature mice to be used in experiments of the Program.
Specific Aim 2. The Core will prepare extracts of RNA and protein from gonads and/or germ cells to be used for expression analyses and RNA-seq experiments of the Program.
Specific Aim 3. The Core will conduct chromatin-immunoprecipitation on gonads or germ cells to support ChlP-seq experiments of the Program. Overall, these aims ensure that all investigators will have the necessary experimental materials to achieve their individual Project goals as well as to collectively achieve the overall goals of the Program.
|Narasimhan, Vagheesh M; Hunt, Karen A; Mason, Dan et al. (2016) Health and population effects of rare gene knockouts in adult humans with related parents. Science 352:474-7|
|Huang, Fang; Sirinakis, George; Allgeyer, Edward S et al. (2016) Ultra-High Resolution 3D Imaging of Whole Cells. Cell 166:1028-40|
|Ball, Robyn L; Fujiwara, Yasuhiro; Sun, Fengyun et al. (2016) Regulatory complexity revealed by integrated cytological and RNA-seq analyses of meiotic substages in mouse spermatocytes. BMC Genomics 17:628|
|Sun, Fengyun; Fujiwara, Yasuhiro; Reinholdt, Laura G et al. (2015) Nuclear localization of PRDM9 and its role in meiotic chromatin modifications and homologous synapsis. Chromosoma 124:397-415|
|Didion, John P; Morgan, Andrew P; Clayshulte, Amelia M-F et al. (2015) A multi-megabase copy number gain causes maternal transmission ratio distortion on mouse chromosome 2. PLoS Genet 11:e1004850|
|Baker, Christopher L; Petkova, Pavlina; Walker, Michael et al. (2015) Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots. PLoS Genet 11:e1005512|
|Baker, Christopher L; Kajita, Shimpei; Walker, Michael et al. (2015) PRDM9 drives evolutionary erosion of hotspots in Mus musculus through haplotype-specific initiation of meiotic recombination. PLoS Genet 11:e1004916|
|Walker, Michael; Billings, Timothy; Baker, Christopher L et al. (2015) Affinity-seq detects genome-wide PRDM9 binding sites and reveals the impact of prior chromatin modifications on mammalian recombination hotspot usage. Epigenetics Chromatin 8:31|
|Baker, Christopher L; Walker, Michael; Kajita, Shimpei et al. (2014) PRDM9 binding organizes hotspot nucleosomes and limits Holliday junction migration. Genome Res 24:724-32|