The Experts below are selected from a list of 16527 Experts worldwide ranked by ideXlab platform
Adam Amsterdam - One of the best experts on this subject based on the ideXlab platform.
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Retroviral-mediated Insertional Mutagenesis in Zebrafish.
Methods in Cell Biology, 2011Co-Authors: Adam Amsterdam, Gaurav Kumar Varshney, Shawn Michael BurgessAbstract:Since the initial publication of this chapter in 2004, additional methodologies have been developed which could improve and/or complement the original retroviral-mediated Insertional Mutagenesis. Retroviral vectors have also been shown to be useful for goals other than Mutagenesis. In addition, retroviral-mediated Insertional Mutagenesis has been applied to zebrafish for use in reverse genetics as well as forward screening. Finally, the Insertional mutant collection described herein has been screened by a number of labs to find a host of mutants (with genes already identified) with developmental and/or growth defects affecting the eye, liver, skin, craniofacial skeleton, kidney, myeloid cells, hematopoietic stem cells, and axon pathfinding, as well as mutants with defects in the cell cycle or DNA damage response, altered aging properties, and modulated cardiac repolarization. The major complementary approaches and new uses of this technique include:
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Insertional Mutagenesis strategies in zebrafish
Genome Biology, 2007Co-Authors: Sridhar Sivasubbu, Adam Amsterdam, Darius Balciunas, Stephen C. EkkerAbstract:We review here some recent developments in the field of Insertional Mutagenesis in zebrafish. We highlight the advantages and limitations of the rich body of retroviral methodologies, and we focus on the mechanisms and concepts of new transposon-based Mutagenesis approaches under development, including prospects for conditional 'gene trapping' and 'gene breaking' approaches.
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Retroviral-mediated Insertional Mutagenesis in zebrafish.
Methods in Cell Biology, 2004Co-Authors: Adam Amsterdam, Nancy HopkinsAbstract:Abstract Since the initial publication of this chapter in 2004, additional methodologies have been developed which could improve and/or complement the original retroviral-mediated Insertional Mutagenesis. Retroviral vectors have also been shown to be useful for goals other than Mutagenesis. In addition, retroviral-mediated Insertional Mutagenesis has been applied to zebrafish for use in reverse genetics as well as forward screening. Finally, the Insertional mutant collection described herein has been screened by a number of labs to find a host of mutants (with genes already identified) with developmental and/or growth defects affecting the eye, liver, skin, craniofacial skeleton, kidney, myeloid cells, hematopoietic stem cells, and axon pathfinding, as well as mutants with defects in the cell cycle or DNA damage response, altered aging properties, and modulated cardiac repolarization. The major complementary approaches and new uses of this technique include: • Pseudotyped retroviruses have been used to deliver enhancer trap vectors, which allows selection of insertions in or near genes with particular expression patterns. Although the mutagenicity of these vectors has yet to be determined, they are useful purely because they generate a large number of transgenic lines with visible reporters (e.g., Green Fluorescent Protein GFP) expressed in interesting patterns and they provide information regarding gene regulation in the context of genomic organization. • Gain-of-function vectors have been designed to allow for dominant genetic screens. Thus, genes whose overexpression results in phenotypes of interest can be efficiently identified. • Retroviruses can be used to make a library of insertions in which hundreds of thousands of mapped insertions can be recovered from frozen sperm samples. Such libraries could serve as on-the-shelf reverse genetic resources, whereby one can obtain a mutation in nearly any gene by simply recovering an insertion in that gene from the frozen sperm, similar to the use of gene-trap insertions and genome-wide gene targeting in ES cells in the mouse. • Transposons have been shown to be nearly as effective transgenesis vectors as retroviruses and thus may be used in similar screens – both for mutagenicity and gene traps and enhancer traps. It is possible that retroviruses and transposons could have different insertion site biases, making them important complementary technologies for genome-wide screening.
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Insertional Mutagenesis in zebrafish
Developmental Dynamics, 2003Co-Authors: Adam AmsterdamAbstract:Insertional Mutagenesis is a method for identifying genes essential for a given biological process by using the integration of DNA as the mutagen, thereby facilitating the cloning of the mutated gene. The use of retrovirus-mediated Insertional Mutagenesis in zebrafish has led to the mutation and rapid identification of hundreds of genes required for embryonic development and cell viability and growth, revealing the diversity of gene products required for the development of this vertebrate. Here, I will review the methodology of this approach and the results to date, as well as other potential ways to use Insertional Mutagenesis for genetic screens.
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A large-scale Insertional Mutagenesis screen in zebrafish.
Genes & Development, 1999Co-Authors: Adam Amsterdam, Shawn Michael Burgess, Gregory Golling, Wenbiao Chen, Zhaoxia Sun, Karen Townsend, Sarah Farrington, Maryann Haldi, Nancy HopkinsAbstract:It is estimated that ~2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5‐7 of age. We are performing a large-scale Insertional Mutagenesis screen in the zebrafish with the goal of isolating ~1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared ~36,000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000‐1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed ~80,000‐150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of Insertional mutagen is 1 embryonic lethal mutation per 70‐100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F5. We screened an estimated 760 insertions among F3 progeny from 92 F2 families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of Mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale Insertional Mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.
Neal G Copeland - One of the best experts on this subject based on the ideXlab platform.
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Transposon Insertional Mutagenesis Models of Cancer
Cold Spring Harbor protocols, 2014Co-Authors: Karen M. Mann, Nancy A Jenkins, Neal G Copeland, Michael B. MannAbstract:Transposon-based Insertional Mutagenesis in the mouse provides a powerful approach for identifying new cancer genes. Transposon insertions in cancer genes are selected during tumor development because of their positive effect on tumor growth, and the transposon insertion sites in tumors thus serve as tags for identifying new cancer genes. Direct comparisons of transposon-mutated genes in mouse tumors with mutated genes in human tumors can lend insight into the genes and signaling pathways that drive tumorigenesis. This is critical for prioritizing genes for further study, either for their efficacy as biomarkers or drug targets. In this article, we will introduce DNA transposon-based systems used for gene discovery in mice and discuss their application to identify candidate cancer genes in light of recently published tumor studies.
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tumor suppressor gene identification using retroviral Insertional Mutagenesis in blm deficient mice
The EMBO Journal, 2006Co-Authors: Takeshi Suzuki, Nancy A Jenkins, Ken Ichi Minehata, Keiko Akagi, Neal G CopelandAbstract:Retroviral Insertional Mutagenesis preferentially identifies oncogenes rather than tumor suppressor (TS) genes, presumably because a single retroviral-induced mutation is sufficient to activate an oncogene and initiate a tumor, whereas two mutations are needed to inactivate a TS gene. Here we show that TS genes can be identified by Insertional Mutagenesis when the screens are performed in Blm-deficient backgrounds. Blm-deficient mice, like Bloom syndrome patients, have increased frequencies of mitotic recombination owing to a mutation in the RecQ protein-like-3 helicase gene. This increased mitotic recombination increases the likelihood that an Insertional mutation in one allele of a TS gene will become homozygoused by non-sister chromatid exchange and the homozygosity of the insertion provides a marker for identifying the TS gene. We also show that known as well as novel TS genes can be identified by Insertional Mutagenesis in Blm-deficient mice and identify two JmjC family proteins that contribute to genome stability in species as evolutionarily diverse as mammals and Caenorhabditis elegans.
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Tumor suppressor gene identification using retroviral Insertional Mutagenesis in Blm‐deficient mice
The EMBO Journal, 2006Co-Authors: Takeshi Suzuki, Nancy A Jenkins, Ken Ichi Minehata, Keiko Akagi, Neal G CopelandAbstract:Retroviral Insertional Mutagenesis preferentially identifies oncogenes rather than tumor suppressor (TS) genes, presumably because a single retroviral-induced mutation is sufficient to activate an oncogene and initiate a tumor, whereas two mutations are needed to inactivate a TS gene. Here we show that TS genes can be identified by Insertional Mutagenesis when the screens are performed in Blm-deficient backgrounds. Blm-deficient mice, like Bloom syndrome patients, have increased frequencies of mitotic recombination owing to a mutation in the RecQ protein-like-3 helicase gene. This increased mitotic recombination increases the likelihood that an Insertional mutation in one allele of a TS gene will become homozygoused by non-sister chromatid exchange and the homozygosity of the insertion provides a marker for identifying the TS gene. We also show that known as well as novel TS genes can be identified by Insertional Mutagenesis in Blm-deficient mice and identify two JmjC family proteins that contribute to genome stability in species as evolutionarily diverse as mammals and Caenorhabditis elegans.
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Insertional Mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells
Blood, 2005Co-Authors: Nancy A Jenkins, Neal G CopelandAbstract:Retroviruses can induce hematopoietic disease via Insertional Mutagenesis of cancer genes and provide valuable molecular tags for cancer gene discovery. Here we show that Insertional Mutagenesis can also identify genes that promote the immortalization of hematopoietic cells, which normally have only limited self-renewal. Transduction of mouse bone marrow cells with replication-incompetent murine stem cell virus (MSCV) expressing only neo, followed by serial passage in liquid culture containing stem cell factor (SCF) and interleukin-3 (IL-3), produced immortalized immature myeloid cell lines with neutrophil and macrophage differentiation potential in about 50% of the infected cultures. More than half of the lines have MSCV insertions at Evi1 or Prdm16. These loci encode transcription factor homologs and are validated human myeloid leukemia genes. Integrations are located in intron 1 or 2, where they promote expression of truncated proteins lacking the PRDI-BF1-RIZ1 homologous (PR) domain, similar to what is observed in human leukemias with EVI1 or PRDM16 mutations. Evi1 overexpression alone appears sufficient to immortalize immature myeloid cells and does not seem to require any other cooperating mutations. Genes identified by Insertional Mutagenesis by their nature could also be involved in immortalization of leukemic stem cells, and thus represent attractive drug targets for treating cancer.
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cooperating cancer gene identification through oncogenic retrovirus induced Insertional Mutagenesis
Blood, 2005Co-Authors: Yang Du, Sally E Spence, Nancy A Jenkins, Neal G CopelandAbstract:Multiple cooperating mutations that deregulate different signaling pathways are required to induce cancer. Identifying these cooperating mutations is a prerequisite for developing better combinatorial therapies for treating cancer. Here we show that cooperating cancer mutations can be identified through oncogenic-retrovirus-induced Insertional Mutagenesis. Among 13 myeloid leukemias induced by transplanting into mice bone marrow cells infected in vitro with a replication-defective retrovirus carrying the Sox4 oncogene, 9 contained Insertional mutations at known or suspected cancer genes. This likely occurred because rare bone marrow cells, in which the oncogenic retrovirus happened to integrate and in which it mutated a cooperating cancer gene, were selected because the host harbored a cooperating cancer mutation. Cooperativity between Sox4 and another gene, Mef2c, was subsequently confirmed in transplantation studies, in which deregulated Mef2c expression was shown to accelerate the myeloid leukemia induced by Sox4. Insertional Mutagenesis of cooperating cancer genes by a defective oncogenic retrovirus provides a new method for identifying cooperating cancer genes and could aid in the development of better therapies for treating cancer. (Blood. 2005;106:2498-2505)
Nancy Hopkins - One of the best experts on this subject based on the ideXlab platform.
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Retroviral-mediated Insertional Mutagenesis in zebrafish.
Methods in Cell Biology, 2004Co-Authors: Adam Amsterdam, Nancy HopkinsAbstract:Abstract Since the initial publication of this chapter in 2004, additional methodologies have been developed which could improve and/or complement the original retroviral-mediated Insertional Mutagenesis. Retroviral vectors have also been shown to be useful for goals other than Mutagenesis. In addition, retroviral-mediated Insertional Mutagenesis has been applied to zebrafish for use in reverse genetics as well as forward screening. Finally, the Insertional mutant collection described herein has been screened by a number of labs to find a host of mutants (with genes already identified) with developmental and/or growth defects affecting the eye, liver, skin, craniofacial skeleton, kidney, myeloid cells, hematopoietic stem cells, and axon pathfinding, as well as mutants with defects in the cell cycle or DNA damage response, altered aging properties, and modulated cardiac repolarization. The major complementary approaches and new uses of this technique include: • Pseudotyped retroviruses have been used to deliver enhancer trap vectors, which allows selection of insertions in or near genes with particular expression patterns. Although the mutagenicity of these vectors has yet to be determined, they are useful purely because they generate a large number of transgenic lines with visible reporters (e.g., Green Fluorescent Protein GFP) expressed in interesting patterns and they provide information regarding gene regulation in the context of genomic organization. • Gain-of-function vectors have been designed to allow for dominant genetic screens. Thus, genes whose overexpression results in phenotypes of interest can be efficiently identified. • Retroviruses can be used to make a library of insertions in which hundreds of thousands of mapped insertions can be recovered from frozen sperm samples. Such libraries could serve as on-the-shelf reverse genetic resources, whereby one can obtain a mutation in nearly any gene by simply recovering an insertion in that gene from the frozen sperm, similar to the use of gene-trap insertions and genome-wide gene targeting in ES cells in the mouse. • Transposons have been shown to be nearly as effective transgenesis vectors as retroviruses and thus may be used in similar screens – both for mutagenicity and gene traps and enhancer traps. It is possible that retroviruses and transposons could have different insertion site biases, making them important complementary technologies for genome-wide screening.
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A large-scale Insertional Mutagenesis screen in zebrafish.
Genes & Development, 1999Co-Authors: Adam Amsterdam, Shawn Michael Burgess, Gregory Golling, Wenbiao Chen, Zhaoxia Sun, Karen Townsend, Sarah Farrington, Maryann Haldi, Nancy HopkinsAbstract:It is estimated that ~2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5‐7 of age. We are performing a large-scale Insertional Mutagenesis screen in the zebrafish with the goal of isolating ~1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared ~36,000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000‐1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed ~80,000‐150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of Insertional mutagen is 1 embryonic lethal mutation per 70‐100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F5. We screened an estimated 760 insertions among F3 progeny from 92 F2 families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of Mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale Insertional Mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.
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A large-scale Insertional Mutagenesis screen in zebrafish.
Genes & development, 1999Co-Authors: Adam Amsterdam, Shawn Michael Burgess, Gregory Golling, Wenbiao Chen, Zhaoxia Sun, Karen Townsend, Sarah Farrington, Maryann Haldi, Nancy HopkinsAbstract:It is estimated that approximately 2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5-7 of age. We are performing a large-scale Insertional Mutagenesis screen in the zebrafish with the goal of isolating approximately 1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared approximately 36, 000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000-1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed approximately 80,000-150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of Insertional mutagen is 1 embryonic lethal mutation per 70-100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F(5). We screened an estimated 760 insertions among F(3) progeny from 92 F(2) families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of Mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale Insertional Mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.
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Insertional Mutagenesis and rapid cloning of essential genes in zebrafish
Nature, 1996Co-Authors: Nicholas Gaiano, Adam Amsterdam, Koichi Kawakami, Miguel L. Allende, Thomas S. Becker, Nancy HopkinsAbstract:Large-scale chemical Mutagenesis screens in zebrafish have led to the isolation of thousands of lethal mutations in genes that are essential for embryonic development. However, the cloning of these mutated genes is difficult at present as it requires positional cloning methods. In Drosophila, chemical Mutagenesis screens were complemented with P-element Insertional Mutagenesis which facilitated the cloning of many genes that had been identified by chemical lesions. To facilitate the cloning of vertebrate genes that are important during embryogenesis, we have developed an Insertional Mutagenesis strategy in zebrafish using a retroviral vector. Here, in a pilot screen of 217 proviral insertions, we obtained three Insertional mutants with embryonic lethal phenotypes, and identified two of the disrupted genes. One of these, no arches, is essential for normal pharyngeal arch development, and is homologous to the recently characterized Drosophila zinc-finger gene, clipper, which encodes a novel type of ribonuclease. As it is easy to generate tens to hundreds of thousands of proviral transgenes in zebrafish, it should now be possible to use this screening method to mutate and then rapidly clone a large number of genes affecting vertebrate developmental and cellular processes.
Alessandro Bulfone - One of the best experts on this subject based on the ideXlab platform.
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lentiviral vector based Insertional Mutagenesis identifies genes associated with liver cancer
Nature Methods, 2013Co-Authors: Marco Ranzani, Daniela Cesana, Cynthia C. Bartholomae, Francesca Sanvito, Mauro Pala, Fabrizio Benedicenti, Pierangela Gallina, Lucia Sergi Sergi, Stefania Merella, Alessandro BulfoneAbstract:Transposons and γ-retroviruses have been efficiently used as Insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells and that hamper the identification of early cancer-driving events among bystander and progression-related events. We developed an Insertional Mutagenesis platform based on lentiviral vectors (LVVs) by which we could efficiently induce hepatocellular carcinoma (HCC) in three different mouse models. By virtue of the LVV's replication-deficient nature and broad genome-wide integration pattern, LVV-based Insertional Mutagenesis allowed identification of four previously unknown liver cancer-associated genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. The newly identified genes are likely to play a role in human cancer because they are upregulated, amplified and/or deleted in human HCCs and can predict clinical outcomes of patients.
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Lentiviral vector–based Insertional Mutagenesis identifies genes associated with liver cancer
Nature Methods, 2013Co-Authors: Marco Ranzani, Daniela Cesana, Cynthia C. Bartholomae, Francesca Sanvito, Mauro Pala, Fabrizio Benedicenti, Pierangela Gallina, Lucia Sergi Sergi, Stefania Merella, Alessandro BulfoneAbstract:Transposons and γ-retroviruses have been efficiently used as Insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells and that hamper the identification of early cancer-driving events among bystander and progression-related events. We developed an Insertional Mutagenesis platform based on lentiviral vectors (LVVs) by which we could efficiently induce hepatocellular carcinoma (HCC) in three different mouse models. By virtue of the LVV's replication-deficient nature and broad genome-wide integration pattern, LVV-based Insertional Mutagenesis allowed identification of four previously unknown liver cancer-associated genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. The newly identified genes are likely to play a role in human cancer because they are upregulated, amplified and/or deleted in human HCCs and can predict clinical outcomes of patients.
Stephen C. Ekker - One of the best experts on this subject based on the ideXlab platform.
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trapping cardiac recessive mutants via expression based Insertional Mutagenesis screening
Circulation Research, 2013Co-Authors: Yonghe Ding, Karl J. Clark, Weibin Liu, Yun Deng, Beninio Jomok, Jingchun Yang, Wei Huang, Tao P Zhong, Xueying Lin, Stephen C. EkkerAbstract:Rationale:Mutagenesis screening is a powerful genetic tool for probing biological mechanisms underlying vertebrate development and human diseases. However, the increased colony management efforts in vertebrates impose a significant challenge for identifying genes affecting a particular organ, such as the heart, especially those exhibiting adult phenotypes on depletion. Objective:We aim to develop a facile approach that streamlines colony management efforts via enriching cardiac mutants, which enables us to screen for adult phenotypes. Methods and Results:The transparency of the zebrafish embryos enabled us to score 67 stable transgenic lines generated from an Insertional Mutagenesis screen using a transposon-based protein trapping vector. Fifteen lines with cardiac monomeric red fluorescent protein reporter expression were identified. We defined the molecular nature for 10 lines and bred them to homozygosity, which led to the identification of 1 embryonic lethal, 1 larval lethal, and 1 adult recessive mut...
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Insertional Mutagenesis strategies in zebrafish
Genome Biology, 2007Co-Authors: Sridhar Sivasubbu, Adam Amsterdam, Darius Balciunas, Stephen C. EkkerAbstract:We review here some recent developments in the field of Insertional Mutagenesis in zebrafish. We highlight the advantages and limitations of the rich body of retroviral methodologies, and we focus on the mechanisms and concepts of new transposon-based Mutagenesis approaches under development, including prospects for conditional 'gene trapping' and 'gene breaking' approaches.