Whole exome sequencing of Rb1 and Trp53 deficient (RP) SCLC mouse model
ABSTRACT: Whole exome sequencing of a cell line derived from an Rb1 and Trp53 genetically engineered mouse model (GEMM) to assess the baseline copy number landscape of the cells prior to experimental modification.
Project description:Myc expression cooperates with Rb1 and Trp53 loss in mouse lungs to generate rapid, aggressive, highly metastatic and neuroendocrine-low tumors which are similar to human variant subset of SCLC with high NEUROD1 expression. Targeted drug screening reveals that mouse and human MYC-driven SCLC are vulnerable to Aurora kinase inhibition in combination with chemotherapy in vivo. Overall design: Tumor formation is induced by infecting the conditional Rb1 fl/fl; Trp53fl/fl, LSL-Myc (T58A) and Rb1 fl/fl; Trp53fl/fl, p130 fl/fl GEMMs with adenoviral vectors containing Cgrp promoter driving Cre recombinase. The tumors were macro-dissected from lungs. RNA was extracted from fresh or flash frozen tumors and subjected to single end RNA sequencing.
Project description:In cells derived from a genetically engineered Rb1 and Trp53 loss mouse model of SCLC (RP) expression of one of the three MYC family members induced from the endogenous locus using CRISPR activation. Cells were first stably transfected with the lenti-MS2-p65-HSF1 activator plasmid. Respective sgRNAs targeting either Myc, Mycl or MYCN were then cloned into the lentiSAMv2 system and transfected separately into the MS2-p65-HSF1 cells using lentiviral delivery.
Project description:Cells lacking Rb1 are deficient in differentiation. Loss of Kdm5a rescues myogenic differentiation, as judged by appearance of morphologically normal myotubes that display expression of late markers of differentiation. In order to better understand how Kdm5a loss rescues differentiation, we induced mouse embryonic fibroblasts (MEFs) of different genotypes to undergo myogenic differentiation and analyzed gene expression changes in wild-type, Kdm5a-/-, Rb1-/- and Kdm5a-/-; Rb1-/- cells. Rb1-/- cells stained single nucleated, did not exhibit morphological changes and increased expression of the myogenic marker MYHC. Except for Rb1-/- cells, all other cells were undergoing successful convertion into aligned multinucleated myotubes and were MYHC-positive. We obtained purified populations of myotubes for the wild-type and Kdm5a-/-; Rb1-/- cells. RNA-seq analysis of gene expression in Rb1 or Kdm5a deficient MEFs that were induced for myogenic differentiation.
Project description:We analyzed miRNA-based shRNA off-target effects by transducing Trp53-/- MEFs at single- and high-copy with six well-characterized, potent and weak Trp53 shRNAs. To advance RNAi therapy for KRAS-mutant cancer, we developed a functionally validated library of siRNAs against RAS pathway genes that minimize off-target effects and enable combination gene silencing at low dose. We developed an in vivo model for real-time tracking of nanoparticle-based siRNA delivery and offer proof-of-principle that siRNA-mediated inhibition of a single gene (KRAS) or combinations of genes (A/B/C-RAF or KRAS+PIK3C-A/B) can impair the growth of KRAS-mutant colorectal cancer xenografts. Trp53-/- MEFs were transduced with LMP expressing Trp53 shRNAs at single copy (11-21% infection efficiency) and high copy (>98% infection efficiency), selected on puromycin and grown in absence of the selection agent before harvest. Uninfected Trp53-/- MEFs and Trp53-/- MEFs infected with an empty vector control served as “no shRNA” reference.
Project description:Transcriptional profilling of 60 day old prostate tissue from Rb1F/F:Trp53F/F:PB-Cre4 or Rb1R654W/F:Trp53F/F:PB-cre4 mice versus wild type prostate tissue. Profiles used to compare deregulation of known E2F target genes in prostate tissue expressing a mutant Rb1 gene to tissue lacking Rb1 expression. Tissue from two Rb1F/F:Trp53F/F:PB-Cre4 (null) mice or two Rb1654/F:Trp53F/F:PB-cre4 (mutant) mice compared to a pool of tissue from 3 wild type mice using two color arrays.
Project description:Loss of Pten in the KrasG12D;Amhr2-Cre mutant mice leads to the transformation of ovarian surface epithelial (OSE) cells and rapid development of low-grade, invasive serous adenocarcinomas. Tumors occur with 100% penetrance and express elevated expression of wild type tumor repressor protein 53 (TRP53). To test the functions of TRP53 in the Pten;Kras (Trp53+) mice, we disrupted the Trp53 gene yielding Pten;Kras(Trp53-) mice. By comparing morphology and gene expression profiles in the Trp53+ and Trp53- OSE cells, we document that wild-type TRP53 acts as a major promoter of OSE cell survival and differentiation: cells lacking Trp53 are transformed yet are less adherent, migratory and invasive and exhibit a gene expression profile more like normal OSE cells. These results provide a new paradigm: wild type TRP53 does not preferentially induce apoptotic or senescent related genes in the Pten;Kras(Trp53+) cancer cells but rather increases genes regulating DNA repair, cell cycle progression and proliferation and decreases putative tumor suppressor genes. However, if TRP53 activity is forced higher by exposure to nutlin-3a (an MDM2 antagonist), TRP53 suppresses DNA repair genes and induces the expression of genes that control cell cycle arrest and apoptosis. Thus, in the Pten;Kras(Trp53+) mutant mouse OSE cells and likely in human TP53+ low grade ovarian cancer cells, wild type TRP53 controls global molecular changes that are dependent on its activation status. These results suggest that activation of TP53 may provide a promising new therapy for managing type I ovarian cancer and other cancers in humans where wild-type TP53 is expressed. A direct comparison of ovarian surface epithelia cells from three different genotype mice
Project description:[original title] Prkar1a haploinsufficiency in mice leads to an overall increase in tumors caused by other genetic defects or chemical induction We investigated the Prkar1a+/- mice when bred within the Rb1+/- or Trp53+/- genetic backgrounds, or treated with a 2-step skin carcinogenesis protocol. Prkar1a+/-Trp53+/- mice developed more bone sarcomas than Trp53+/- mice (p<0.05) and Prkar1a+/-Rb1+/- mice grew more and larger pituitary and thyroid tumors than Rb1+/- mice. All mice with double heterozygosity had significantly reduced life-spans compared with their single-heterozygous counterparts. Finally, Prkar1a+/- mice developed more papillomas than wild-type animals when treated with a 2-step skin carcinogenesis protocol. A whole-genome transcriptome profiling of tumors produced by all three models identified Wnt signaling as the main pathway activated by abnormal cAMP signaling in these tissues, along with (expected) cell cycle gene abnormalities, all confirmed by qRT-PCR array and immunohistochemical analyses. Overall design: Total RNA obtained from skin papillomas from Prkar1a+/- mice were compared to those obtained from WT mice. Total RNA obtained from pituitary and thyroid tumors from Rb1+/- mice were compared to those samples obtained from Prkar1a+/- Rb1+/- mice. Total RNA obtained from sarcomas from Tp53+/- mice were compared to those samples obtained from Prkar1a+/- Tp53+/- mice.
Project description:Small Cell Lung Cancer (SCLC) is the most aggressive type of lung cancer with early metastatic dissemination and invariable development of resistant disease for which no effective treatment is available to date. Mouse models of SCLC based on inactivation of Rb1 and Trp53 developed earlier showed frequent amplifications of two transcription factor genes: Nfib and Mycl. Overexpression of Nfib but not Mycl in SCLC mouse results in an enhanced and altered metastatic profile, and appears to be associated with genomic instability. NFIB promotes tumor heterogeneity with the concomitant expansive growth of poorly differentiated, highly proliferative, and invasive tumor cell populations. Consistent with the mouse data, NFIB expression in high-grade human neuroendocrine carcinomas correlates with advanced stage III/IV disease warranting its further assessment as a potentially valuable progression marker in a clinical setting. Genomic DNA from mouse small cell lung tumor samples was analyzed by mate pair sequencing and low coverage sequencing. And RNA from Nfib overexpressing mouse small cell lung cancer cell lines was further analyzed for high quality RNA profiles using Illumina Hiseq2500. This series contains only RNA-seq data.
Project description:Background: Whole exome sequencing (WES) has been proven to serve as a valuable basis for various applications such as variant calling and copy number variation (CNV) analyses. For those analyses the read coverage should be optimally balanced throughout protein coding regions at sufficient read depth. Unfortunately, WES is known for its uneven coverage within coding regions due to GC-rich regions or off-target enrichment. Results: In order to examine the irregularities of WES within genes, we applied Agilent SureSelectXT exome capture on human samples and sequenced these via Illumina in 2x101 paired-end mode. As we suspected the sequenced insert length to be crucial in the uneven coverage of exome captured samples, we sheared 12 genomic DNA samples to two different DNA insert size lengths, namely 130 and 170 bp. Interestingly, although mean coverages of target regions were clearly higher in samples of 130 bp insert length, the level of evenness was more pronounced in 170 bp samples. Moreover, merging overlapping paired-end reads revealed a positive effect on evenness indicating overlapping reads as another reason for the unevenness. In addition, mutation analysis on a subset of the samples was performed. In these isogenic subclones almost twofold mutations were failed in the 130 bp samples when compared to the 170 bp samples. Visual inspection of the discarded mutation sites exposed low coverages at the sites embedded in high amplitudes of coverage depth in the affected region. Conclusions: Producing longer insert reads could be a good strategy to achieve better uniform read coverage in coding regions and hereby enhancing the effective sequencing yield to provide an improved basis for further variant calling and CNV analyses.
Project description:Retinoblastoma gene (Rb1) is required for proper cell cycle exit in the developing mouse inner ear and its deletion in the embryo leads to proliferation of sensory progenitor cells that differentiate into hair cells and supporting cells. In the Pou4f3-Cre:Rb1 flox/flox (Rb1 cKO) inner ear, utricular hair cells differentiate and survive into adulthood whereas differentiation and survival of cochlear hair cells are impaired. To comprehensively survey the pRb pathway in the mammalian inner ear, we performed microarray analysis of Rb1 cKO cochlea and utricle. P6 or 2-month control and Rb1 cKO littermates were euthanized and the inner ear tissues were dissected. Total RNA was extracted from the pooled samples. Technical duplicates of the pooled RNA were used for microarray.