Project description:Transcriptional profiling of FACS-sorted ureteric bud-derivative cells from Hoxb7CreGFP mice without floxed Bdkrb2 allele (WT) or with homozygously floxed Bdkrb2 alleles (MUT) at the age of postnatal one. Both WT and MUT mice have R26RTdTomato allele as a recombinase efficiency indicator, which allows the FACS to sort cells according to both GFP and TdTomato. This experiment aimed to uncover the genome-wide alternation in gene expression resulting from the removal of Bdkrb2 in the ureteric buds and their derivative collecting ducts.
Project description:Wnt9b is expressed in the ureteric bud of the kidney at all stages of development. In Wnt9b mutants, the ureteric bud forms but the metanephric mesenchyme is never induced to undergo differentiation. We used microarrays to compare differences in gene expression between wildtype and Wnt9b mutants. To focus on direct targets, mesenchymes isolated away from the E11.5 ureteric bud were assayed
Project description:E11.5 metanephric mesenchyme and ureteric bud were dissected from the E11.5 kidney rudiment using fine manual microdissection (ureteric bud only) or both fine manual microdissection and laser capture microdissection (metanephric mesenchyme) to define the gene expression profiles of these structures. Additionally, HoxA11, HoxD11 compound null E11.5 metanephric mesenchyme was obtained through laser capture microdissection allowing analysis of possible Hox targets in kidney development. Targets from multiple biological replicates of each were generated and the expression profiles were determined using Affymetrix MOE430_v2 arrays. Using microdissection techniques, ureteric bud and metanephric mesenchyme were dissected from E11.5 kidney rudiments allowing the identificated genes specifically regulated in either structure. In addition, Hoxa11, Hoxd11 compound null E11.5 metanephric mesenchyme were normalized to wild type embryonic controls allowing the identification of potential Hox targets in normal kidney development. Each structure/genotype were represented in biological (seperate embryo) replicate.
Project description:The long term objective is to create an encyclopedia of the expression levels of all genes in multiple components of the developing kidney. The central thesis is straightforward. The combination of fluorescent activated cell sorting (FACS) plus microarray analysis offers a powerful, efficient and effective method for the creation of a global gene expression atlas of the developing kidney. Microarrays with essentially complete genome coverage can be used to quantitate expression levels of every gene in FACS isolated components of the developing kidney. The ensuing rapid read-out provides an expression atlas that is more sensitive, more economical and more complete than would be possible by in situ hybridizations alone. HoxB7-GFP transgenic mice were utilized to isolate the ureteric bud cells from E10.5 embryonic kidneys. The ureteric bud cells were isolated from embryos using microdissection, trypsin treatment and FACS. The RNA was isolated from purified ureteric bud cells and the gene expression profiles were determined by microarrays.
Project description:E11.5 metanephric mesenchyme and ureteric bud were dissected from the E11.5 kidney rudiment using fine manual microdissection (ureteric bud only) or both fine manual microdissection and laser capture microdissection (metanephric mesenchyme) to define the gene expression profiles of these structures. Additionally, HoxA11, HoxD11 compound null E11.5 metanephric mesenchyme was obtained through laser capture microdissection allowing analysis of possible Hox targets in kidney development. Targets from multiple biological replicates of each were generated and the expression profiles were determined using Affymetrix MOE430_v2 arrays. Keywords: embryonic metanephric kidney, kidney development, Hoxa11, Hoxd11, compound null targeted mice
Project description:BRCA1 nestin CRE conditional knockout cortrices of P7 animals were compared to wildtype littermates to characterize the mutant phenotype. Keywords: expression BRCA1 conditional knockouts using nestin CRE and a null allele with an inverted neo cassette at the 5' end of the exon 11 of BRCA1 on one floxed allele flanking exons 5-13. Cortices of 3 wildtype animals were compred to 3 BRCA cKO at postnatal day 7.
Project description:Grb2 is a small SH2-SH3 adaptor molecule that interacts with activated tyrosine kinase receptors (RTKs), providing a crucial link towards downstream activation of pro-proliferative and pro-survival ERK and Akt signaling pathways. Ret and FGFR2, two RTKs that interact with Grb2, play important roles in ureteric branching and the proper establishment of the renal collecting duct system, but it is unclear whether Grb2 is required in this process. In this study, we selectively ablated a conditional floxed allele of the Grb2 gene within the ureteric epithelial lineage in mice and demonstrate that Grb2 signaling is essentially required for proper collecting duct development. Ureteric Grb2 deficiency results in perinatal lethality and severe renal hypodysplasia closely reminiscent of the rudimentary kidney phenotypes observed in Ret-null mutant mice. Grb2 loss attenuates ERK and Akt activation in the ureteric epithelia resulting in pronounced impairment of ureteric branching. Gene expression analysis reveals that Grb2 deficiency results in defective induction of genes implicated in both ureteric branching and reciprocal mesenchymal metanephric induction. Our findings therefore strongly indicate that Grb2 is a physiologically-relevant major RTK signaling relay partner that promotes renal branching morphogenesis and the proper development of the collecting duct system and the urogenital tract. Microarray was used to profile and compare the transcriptomes of developing kidneys of E14.5 Grb2 conditional knockout (ureteric-bud specific) and wild-type embryos
Project description:We aimed to define epithelial-specific genes in the kidney. In the developing mouse kidney at E12.5 epithelial cells are restricted to the ureteric bud, while mesenchymal cells surrounding the ureteric bud are non-epithelial. The mouse renal epithelial cell line mIMCD-3 was used to represent kidney epithelia in vitro. Gene expression was analyzed using Affymetrix microarrays in ureteric bud stalks, ureteric bud tips, and mIMCD-3 cells and compared to metanephric mesenchyme.