Modeling sporadic loss of heterozygosity in mice by using mosaic analysis with double markers (MADM).
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ABSTRACT:
SUBMITTER: Muzumdar MD
PROVIDER: S-EPMC1810340 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mosaic Analysis with Double Markers (MADM) is a method for generating genetically mosaic mice, in which sibling mutant and wild-type cells are labeled with different fluorescent markers. It is a powerful tool that enables analysis of gene function at the single cell level in vivo. It requires transgenic cassettes to be located between the centromere and the mutation in the gene of interest on the same chromosome. Here we compare procedures for introduction of MADM cassettes into new loci in the mouse genome, and describe new approaches for expanding the utility of MADM. We show that: 1) Targeted homologous recombination outperforms random transgenesis in generation of reliably expressed MADM cassettes, 2) MADM cassettes in new genomic loci need to be validated for biallelic and ubiquitous expression, 3) Recombination between MADM cassettes on different chromosomes can be used to study reciprocal chromosomal deletions/duplications, and 4) MADM can be modified to permit transgene expression by combining it with a binary expression system. The advances described in this study expand current, and enable new and more versatile applications of MADM.
| S-EPMC3314016 | biostudies-literature
Project description:Mosaic analysis with double markers (MADM) offers one approach to visualize and concomitantly manipulate genetically defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage, single-cell morphology and physiology, genomic imprinting phenotypes, and dissection of cell-autonomous gene functions in vivo in health and disease. Yet, MADM can only be applied to <25% of all mouse genes on select chromosomes to date. To overcome this limitation, we generate transgenic mice with knocked-in MADM cassettes near the centromeres of all 19 autosomes and validate their use across organs. With this resource, >96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond a proof of principle, we apply our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We find striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division.
| S-EPMC8317686 | biostudies-literature
Project description:Mosaic Analysis with Double Markers (MADM) is a mouse genetic system that allows simultaneous gene knockout and fluorescent labeling of sparse, clonally-related cells within an otherwise normal mouse, thereby circumventing embryonic lethality problems and providing single-cell resolution for phenotypic analysis in vivo. The clonal efficiency of MADM is intrinsically low because it relies on Cre/loxP-mediated mitotic recombination between two homologous chromosomes rather than within the same chromosome, as in the case of conditional knockout (CKO). Although sparse labeling enhances in vivo resolution, the original MADM labels too few or even no cells when a low-expressing Cre transgene is used or a small population of cells is studied. Recently, we described the usage of a new system, MADM-ML, which contains three mutually exclusive, self-recognizing loxP variant sites as opposed to a single loxP site present in the original MADM system (referred to as MADM-SL in this paper). Here we carefully compared the recombination efficiency between MADM-SL and MADM-ML using the same Cre transgene, and found that the new system labels significantly more cells than the original system does. When we established mouse medulloblastoma models with both the original and the new MADM systems, we found that, while the MADM-SL model suffered from varied tumor progression and incomplete penetrance, the MADM-ML model had consistent tumor progression and full penetrance of tumor formation. Therefore MADM-ML, with its higher recombination efficiency, will broaden the applicability of MADM for studying many biological questions including normal development and disease modeling at cellular resolution in vivo.
| S-EPMC3797059 | biostudies-literature
Project description:PURPOSE:Wilms' tumor is a childhood cancer of the kidney with an incidence of approximately 1 in 10,000. Cooccurrence of Wilms' tumor with 2q37 deletion syndrome, an uncommon constitutional chromosome abnormality, has been reported previously in three children. Given these are independently rare clinical entities, we hypothesized that 2q37 harbors a tumor suppressor gene important in Wilms' tumor pathogenesis. EXPERIMENTAL DESIGN:To test this, we performed loss of heterozygosity analysis in a panel of 226 sporadic Wilms' tumor samples and mutation analysis of candidate genes. RESULTS:Loss of heterozygosity was present in at least 4% of cases. Two tumors harbored homozygous deletions at 2q37.1, supporting the presence of a tumor suppressor gene that follows a classic two-hit model. However, no other evidence of second mutations was found, suggesting that heterozygous deletion alone may be sufficient to promote tumorigenesis in concert with other genomic abnormalities. We show that miR-562, a microRNA within the candidate region, is expressed only in kidney and colon and regulates EYA1, a critical gene for renal development. miR-562 expression is reduced in Wilms' tumor and may contribute to tumorigenesis by deregulating EYA1. Two other candidate regions were localized at 2q37.3 and 2qter, but available data from patients with constitutional deletions suggest that these probably do not confer a high risk for Wilms' tumor. CONCLUSIONS:Our data support the presence of a tumor suppressor gene at 2q37.1 and suggest that, in individuals with constitutional 2q37 deletions, any increased risk for developing Wilms' tumor likely correlates with deletions encompassing 2q37.1.
| S-EPMC2756455 | biostudies-literature
Project description:BackgroundLoss of heterozygosity (LOH) is an important marker for one of the 'two-hits' required for tumor suppressor gene inactivation. Traditional methods for mapping LOH regions require the comparison of both tumor and patient-matched normal DNA samples. However, for many archival samples, patient-matched normal DNA is not available leading to the under-utilization of this important resource in LOH studies. Here we describe a new method for LOH analysis that relies on the genome-wide comparison of heterozygosity of single nucleotide polymorphisms (SNPs) between cohorts of cases and un-matched healthy control samples. Regions of LOH are defined by consistent decreases in heterozygosity across a genetic region in the case cohort compared to the control cohort.MethodsDNA was collected from 20 Follicular Lymphoma (FL) tumor samples, 20 Diffuse Large B-cell Lymphoma (DLBCL) tumor samples, neoplastic B-cells of 10 B-cell Chronic Lymphocytic Leukemia (B-CLL) patients and Buccal cell samples matched to 4 of these B-CLL patients. The cohort heterozygosity comparison method was developed and validated using LOH derived in a small cohort of B-CLL by traditional comparisons of tumor and normal DNA samples, and compared to the only alternative method for LOH analysis without patient matched controls. LOH candidate regions were then generated for enlarged cohorts of B-CLL, FL and DLBCL samples using our cohort heterozygosity comparison method in order to evaluate potential LOH candidate regions in these non-Hodgkin's lymphoma tumor subtypes.ResultsUsing a small cohort of B-CLL samples with patient-matched normal DNA we have validated the utility of this method and shown that it displays more accuracy and sensitivity in detecting LOH candidate regions compared to the only alternative method, the Hidden Markov Model (HMM) method. Subsequently, using B-CLL, FL and DLBCL tumor samples we have utilised cohort heterozygosity comparisons to localise LOH candidate regions in these subtypes of non-Hodgkin's lymphoma. Detected LOH regions included both previously described regions of LOH as well as novel genomic candidate regions.ConclusionsWe have proven the efficacy of the use of cohort heterozygosity comparisons for genome-wide mapping of LOH and shown it to be in many ways superior to the HMM method. Additionally, the use of this method to analyse SNP microarray data from 3 common forms of non-Hodgkin's lymphoma yielded interesting tumor suppressor gene candidates, including the ETV3 gene that was highlighted in both B-CLL and FL.
| S-EPMC2885361 | biostudies-literature
Project description:Osteochondromas occur as sporadic solitary lesions or as multiple lesions, characterizing the hereditary multiple exostoses syndrome (EXT). Approximately 15% of all chondrosarcomas arise within the cartilaginous cap of an osteochondroma. EXT is genetically heterogeneous, and two genes, EXT1 and EXT2, located on 8q24 and 11p11-p12, respectively, have been cloned. It is still unclear whether osteochondroma is a developmental disorder or a true neoplasm. Furthermore, it is unclear whether inactivation of both alleles of an EXT gene, according to the tumor-suppressor model, is required for osteochondroma development, or whether a single EXT germline mutation acts in a dominant negative way. We therefore studied loss of heterozygosity and DNA ploidy in eight sporadic and six hereditary osteochondromas. EXT1- and EXT2-mutation analysis was performed in a total of 34 sporadic and hereditary osteochondromas and secondary peripheral chondrosarcomas. We demonstrated osteochondroma to be a true neoplasm, since aneuploidy was found in 4 of 10 osteochondromas. Furthermore, LOH was almost exclusively found at the EXT1 locus in 5 of 14 osteochondromas. Four novel constitutional cDNA alterations were detected in exon 1 of EXT1. Two patients with multiple osteochondromas demonstrated a germline mutation combined with loss of the remaining wild-type allele in three osteochondromas, indicating that, in cartilaginous cells of the growth plate, inactivation of both copies of the EXT1 gene is required for osteochondroma formation in hereditary cases. In contrast, no somatic EXT1 cDNA alterations were found in sporadic osteochondromas. No mutations were found in the EXT2 gene.
| S-EPMC1377975 | biostudies-other
Project description:BackgroundHybridization, genetic mixture of distinct populations, gives rise to myriad recombinant genotypes. Characterizing the genomic composition of hybrids is critical for studies of hybrid zone dynamics, inheritance of traits, and consequences of hybridization for evolution and conservation. Hybrid genomes are often summarized either by an estimate of the proportion of alleles coming from each ancestral population or classification into discrete categories like F1, F2, backcross, or merely "hybrid" vs. "pure". In most cases, it is not realistic to classify individuals into the restricted set of classes produced in the first two generations of admixture. However, the continuous ancestry index misses an important dimension of the genotype. Joint consideration of ancestry together with interclass heterozygosity (proportion of loci with alleles from both ancestral populations) captures all of the information in the discrete classification without the unrealistic assumption that only two generations of admixture have transpired.MethodsI describe a maximum likelihood method for joint estimation of ancestry and interclass heterozygosity. I present two worked examples illustrating the value of the approach for describing variation among hybrid populations and evaluating the validity of the assumption underlying discrete classification.ResultsNaively classifying natural hybrids into the standard six line cross categories can be misleading, and false classification can be a serious problem for datasets with few molecular markers. My analysis underscores previous work showing that many (50 or more) ancestry informative markers are needed to avoid erroneous classification.ConclusionAlthough classification of hybrids might often be misleading, valuable inferences can be obtained by focusing directly on distributions of ancestry and heterozygosity. Estimating and visualizing the joint distribution of ancestry and interclass heterozygosity is an effective way to compare the genetic structure of hybrid populations and these estimates can be used in classic quantitative genetic methods for assessing additive, dominant, and epistatic genetic effects on hybrid phenotypes and fitness. The methods are implemented in a freely available package "HIest" for the R statistical software ( http://cran.r-project.org/web/packages/HIest/index.html).
| S-EPMC3572440 | biostudies-literature
Project description:Cancer results from molecular mutations occurring in specific cell types, thus determining the cell-of-origin is critical for effective cancer treatment. Inferring such information from terminal tumors can be misleading because malignant tumor cells tend to acquire aberrant properties. Animal models are widely used because one can initiate mutations in specific cell types. However, cell-of-mutation that harbors initial molecular changes may not directly transform, rather merely passes along mutations to its progeny cell lineage, which then serves as cell-of-origin and finally transforms into malignancy. Such a problem is exemplified in the glioma field: glioma can be induced either by mutating tumor suppressor genes in neural stem cells (NSCs) or by over-expressing oncogenes in restricted progenitor cells such as oligodendrocyte precursor cells (OPCs). However, for the NSC glioma model, it remains unknown whether NSCs directly transform or simply pass along mutations to OPC lineage for malignancy. Here we use a genetic system termed Mosaic Analysis with Double Markers (MADM) to study the earliest stage of gliomagenesis from mutated NSCs. At an in vivo single-cell resolution, we unbiasedly analyzed tumorigenic potential of NSCs and all cell lineages derived from them. At pathologically undetectable pre-transforming phases, we found no significant aberrant growth of mutant NSCs and their derivatives except for the OPC lineage. We then tracked the tumor progression from earliest stage and confirmed the expansion of OPCs lead to gliomagenesis. Consistently, transcriptome profiling reveals that terminal-stage tumor cells display salient OPC features and resemble human proneural subtype of glioblastoma multiform (GBM). Most importantly, introducing the same mutations directly into OPCs was sufficient for malignant transformation and these tumor cells are indistinguishable in their gene expression profiles from those in which the first mutations were introduced into NSCs. Our findings strongly implicate OPCs as the transforming cell type for glioma even when initial mutations could occur in NSCs, and highlight the importance of analyzing early phases of tumorigenesis to distinguish cell-of-origin from cell-of-mutation.
2011-07-01 | GSE26676 | GEO
Project description:Microsatellite instability (MSI) is a mutator phenotype that results from a defective mismatch repair (MMR) pathway. The present study examined the incidence of MSI and loss of heterozygosity (LOH) according to five markers from the panel of the National Cancer Institute (NCI) in 38 colorectal cancer (CRC) patients from the United Arab Emirates (UAE). MSI and LOH were analyzed using fragment analyses in a multiplex PCR setting on a capillary array electrophoresis platform. The expression of the MMR proteins, hMLH1 and hMSH2, was analyzed using immunohistochemistry. The cohort consisted of 17 females (44.7%) and 21 males (55.3%) with mean ages of 59.9 and 63.3 years, respectively. The overall MSI incidence was 31.3% (95% CI, 16.1-50.0), and included three patients with high MSI (MSI-H; 9.4%; 95% CI, 2.0-25.0) and seven patients with low MSI (MSI-L; 21.9%; 95% CI, 10.7-39). LOH was detected in three patients, while the remaining 25 patients (65.8%) showed no instability and were therefore classified as microsatellite stable (MSS). MSI was detected in the following screened markers: Bat25 in seven patients, Bat26 in three patients, adenomatous polyposis coli (APC; D5S346) in five patients, AFM093xh3 (D2S123) in two patients and Mfd15 (D17S250) in three patients. Of the five MSI-positive patients, four (80%) were evidently younger, aged 38, 48, 49 and 59 years, respectively. The MSI-H incidence (9.4%) was lower compared with that of other ethnic groups. In terms of the MMR proteins, hMLH1 expression was deficient in seven patients, of whom three were MSI-H patients, and hMSH2 was deficient in three patients. Fisher's exact test showed significant associations between hMLH1 and MSI when classified as MSS, MSI-L or MSI-H (P=0.0003). No such association was observed with abnormal MMR protein expression, age, cancer stage or gender.
| S-EPMC3813818 | biostudies-other
Project description:ObjectivesVariation in microsatellite sequences that are dispersed in the genome has been linked to a deficiency in cellular mismatch repair system and defects in several genes of this system are involved in carcinogenesis. Our aim in this study was to illustrate microsatellite DNA alteration in esophageal cancer.Materials and methodsDNA was extracted from formalin fixed paraffin embedded (FFPE) tissues from surgical and matched margin-normal samples. Microsatellite instability (MSI) and loss of heterozygosity (LOH) were studied in 50 cases of esophageal squamous cell carcinoma (ESCC) by amplifying six microsatellite markers: D13S260 (13q12.3), D13S267 (13q12.3), D9S171 (9p21), D2S123 (2p), D5S2501 (5q21) and TP53 (17p13.1) analyzed on 6% denaturing polyacrylamide gel electrophoresis.ResultsStatistical analysis indicated a near significant reverse correlation between grade and LOH (P= 0.068, correlation coefficient= -0.272). Specifically, increased LOH in tumor DNA has a significant correlation with increased differentiation from poorly differentiated to well differentiated tumors (P= 0.002 and P= 0.016 respectively). In addition, higher number of chromosomal loci with LOH showed a reverse correlation with lymph node metastasis (P= 0.026, correlation coefficient= -0.485). Furthermore, there was a positive correlation between addiction and MSI (P= 0.026, correlation coefficient= 0.465).ConclusionMicrosatellite DNA alterations may be a prognostic tool for detection and the evolution of prognosis in patients with SCC of esophagus. It can be concluded that regional lymph node metastasis would be less likely with increased heterozygote loci and addiction with any of opium, cigarette, water pipe or alcohol can be a susceptibility factor(s) for MSI.
| S-EPMC5010844 | biostudies-literature