Project description:The CCAAT enhancer binding protein alpha (C/EBPalpha) transcription factor plays a key role in the regulation of growth and differentiation of the granulocytic lineage in the hematopoietic system and CEBPA (encoding C/EBPalpha) is often mutated or deregulated in AML patients. Consistently, mice lacking C/EBPalpha have no mature neutrophils and die within a few hours. However, homozygous knockin mice in which wild type Cebpa has been replaced with a mutant allele (BRM2), which abolish the growth-repressing ability of C/EBPalpha, are viable, but at 8 weeks of age they display myeloid dysplasia with absence of neutrophil granulocytes. Strikingly, in older CebpaBRM2/BRM2 knockin mice the myeloid dysplastic phenotype progress into other myeloid malignancies such as myeloid proliferative syndrome and acute myeloid leukemia-like malignancy. This strongly suggests that secondary mutations in other loci must occur when developing leukemia in the CebpaBRM2/BRM2. In order to identify genes that cooperate with Cebpa mutations in the development of leukemia in CebpaBRM2/BRM2 mice a so-called retroviral insertion mutagenesis screen was performed. Inbred newborn CebpaBRM2/BRM2 and wildtype mice were injected with SRS19-6 retrovirus, which incorporated into the genome and resulted in activation of oncogenes and leukemic progression. When leukemia was evident the mice were euthanized and analyzed. As expected the CebpaBRM2/BRM2 mice had a shorter latency than wildtype mice (186 vs. 276 days). The mice had enlarged spleen, thymus, and/or lymph nodes and were thoroughly investigated by histology, flow cytometry and southern in order to determine the leukemic phenotypes. Most of the CebpaBRM2/BRM2 mice developed an AML-like phenotype, whereas T cell leukemias were most prominent in wildtype mice, showing that mutating Cebpa specifically directs leukemic progression towards a myeloid direction. Genomic instability was analyzed by CGH. Finally, the retroviral insertion loci were identified through a splinkerette-aided PCR strategy. This led to the identification of several novel putative and previously unexplored oncogenes, which might collaborate with mutated Cebpa in the development of AML. Keywords: Disease state analysis In order to identify genes that cooperate with Cebpa mutations in the development of leukemia in CebpaBRM2/BRM2 mice a so-called retroviral insertion mutagenesis screen was performed. Inbred newborn CebpaBRM2/BRM2 and wildtype mice were injected with SRS19-6 retrovirus, which incorporated into the genome and resulted in activation of oncogenes and leukemic progression. When leukemia was evident the mice were euthanized and analyzed. GDNA from tumour tissues were analyzed by CGH using tail gDNA as reference.

Project description:Background We have developed and validated a set of complementary retroviruses that creates a wide range of nested chromosomal deletions. When applied to mouse ES cells, this retrovirus-based method generated deletions ranging from 6 kb to 23 Mb (average 2.9 Mb), with an efficiency of 64% for drugs-selected clones. Importantly, several of engineered ES cell clones, mostly those with large deletions, showed major alteration in cell fate. The set of complementary replication-defective retroviruses exploited the Cre-loxP recombination system and reconstitution of a functional neomycin cassette for selection of recombination events. The first loxP sequenced was delivered using the anchor virus A1. The integration was selected on puromycin. ES cell clones containing one copy of the virus A1 were isolated and expanded (primary clones). A second loxP was introduced by retroviral gene transfer using the saturation virus S1, selected on hygromycin (secondary clones). The transient expression of the recombinase Cre in secondary clones allowed the recombination between the loxP sites and the functional reconstitution of a split neomycin expression cassette. Neomycin resistant clones (tertiary clones) were isolated and expanded. Chromosomal deletions are expected to have occurred in neomycin resistant clones that have lost both puromycin and hygromycin resistance genes. In addition, other chromosomal rearrangements (e.g., inversions, translocations, etc.) are achievable using this system. Aim Inverse-PCR, array-based comparative genomic hybridization (aCGH) and spectral karyotyping were employed to confirm deletions (or other Cre-induced rearrangements) in several tertiary clones and to assess the genomic integrity of the ES cells altered. Conclusions aCGH conclusions are summarized together with complementary results from inverse-PCR and spectral karyotyping in 3 tables: Table 1: Summary of the virus A1 integration Table 2: Characteristics of independent deletions confirmed by IPCR-aCGH Table 3: Confirmed or suspected recombination events in trans Notes for Table 2: Mapping and deletion analyses were done using the UCSC Genome Browser (NCBI mouse Build 33). {a} Tertiary clones are labeled according to their family number (same integration of virus A1), followed by a specific id number. If more than one clone presented a redundant rearrangement within the same group infected with virus S1, only one is reported for clarity. {b} Anomaly that was not present in the primary clone from which the tertiary clone was derived, as determined by aCGH or SKY. ( -), no anomaly; (+), additional anomaly. {c} The deletion is not observed, in agreement with the resolution of aCGH. {d} Normal except for the loss of chromosome Y. {e} Amplification of chromosome 1. {f} Amplification of chromosome 8. {g} Many chromosomes were lost according to SKY. {h} Amplification on chromosome 14. Id, identification; kb, kilobase pairs; no., number; aCGH, array-based comparative genomic hybridization; SKY, spectral karyotyping; I-PCR, inverse-PCR; n.d., not determined. Keywords: comparative genomic hybridization

Project description:The CCAAT enhancer binding protein alpha (C/EBPalpha) transcription factor plays a key role in the regulation of growth and differentiation of the granulocytic lineage in the hematopoietic system and CEBPA (encoding C/EBPalpha) is often mutated or deregulated in AML patients. Consistently, mice lacking C/EBPalpha have no mature neutrophils and die within a few hours. However, homozygous knockin mice in which wild type Cebpa has been replaced with a mutant allele (BRM2), which abolish the growth-repressing ability of C/EBPalpha, are viable, but at 8 weeks of age they display myeloid dysplasia with absence of neutrophil granulocytes. Strikingly, in older CebpaBRM2/BRM2 knockin mice the myeloid dysplastic phenotype progress into other myeloid malignancies such as myeloid proliferative syndrome and acute myeloid leukemia-like malignancy. This strongly suggests that secondary mutations in other loci must occur when developing leukemia in the CebpaBRM2/BRM2. In order to identify genes that cooperate with Cebpa mutations in the development of leukemia in CebpaBRM2/BRM2 mice a so-called retroviral insertion mutagenesis screen was performed. Inbred newborn CebpaBRM2/BRM2 and wildtype mice were injected with SRS19-6 retrovirus, which incorporated into the genome and resulted in activation of oncogenes and leukemic progression. When leukemia was evident the mice were euthanized and analyzed. As expected the CebpaBRM2/BRM2 mice had a shorter latency than wildtype mice (186 vs. 276 days). The mice had enlarged spleen, thymus, and/or lymph nodes and were thoroughly investigated by histology, flow cytometry and southern in order to determine the leukemic phenotypes. Most of the CebpaBRM2/BRM2 mice developed an AML-like phenotype, whereas T cell leukemias were most prominent in wildtype mice, showing that mutating Cebpa specifically directs leukemic progression towards a myeloid direction. Genomic instability was analyzed by CGH. Finally, the retroviral insertion loci were identified through a splinkerette-aided PCR strategy. This led to the identification of several novel putative and previously unexplored oncogenes, which might collaborate with mutated Cebpa in the development of AML. Keywords: Disease state analysis

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Retroviral mutagenesis in haploid ES cells

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