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Journal articles on the topic 'Splinkerette'

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Published: 14 March 2023

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1

Hengen, P. "Vectorette, splinkerette and boomerang DNA amplification." Trends in Biochemical Sciences 20, no. 9 (September 1995): 372–73. http://dx.doi.org/10.1016/s0968-0004(00)89079-9.

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2

Potter, Christopher J., and Liqun Luo. "Splinkerette PCR for Mapping Transposable Elements in Drosophila." PLoS ONE 5, no. 4 (April 13, 2010): e10168. http://dx.doi.org/10.1371/journal.pone.0010168.

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3

Horn, Carsten, Jens Hansen, Frank Schnütgen, Claudia Seisenberger, Thomas Floss, Markus Irgang, Silke De-Zolt, Wolfgang Wurst, Harald von Melchner, and Patricia Ruiz Noppinger. "Splinkerette PCR for more efficient characterization of gene trap events." Nature Genetics 39, no. 8 (August 2007): 933–34. http://dx.doi.org/10.1038/ng0807-933.

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4

Horn, Carsten, Jens Hansen, Frank Schnütgen, Claudia Seisenberger, Thomas Floss, Markus Irgang, Silke De-Zolt, Wolfgang Wurst, Harald von Melchner, and Patricia Ruiz Noppinger. "Erratum: Splinkerette PCR for more efficient characterization of gene trap events." Nature Genetics 39, no. 12 (December 2007): 1528. http://dx.doi.org/10.1038/ng1207-1528.

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5

Cavagnaro, Pablo F., Douglas Senalik, and Philipp W. Simon. "SplinkBES: a splinkerette-based method for generating long end sequences from large insert DNA libraries." BioTechniques 47, no. 2 (August 2009): 681–90. http://dx.doi.org/10.2144/000113122.

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6

Uren, Anthony G., Harald Mikkers, Jaap Kool, Louise van der Weyden, Anders H. Lund, Catherine H. Wilson, Richard Rance, et al. "A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites." Nature Protocols 4, no. 5 (April 30, 2009): 789–98. http://dx.doi.org/10.1038/nprot.2009.64.

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7

Too, W. C. See, Y. C. Liew, and L. L. Few. "Cloning of glyceraldehyde-3-phosphate dehydrogenase from an Antarctic psychrophilic bacterium by inverse and splinkerette PCR." Journal of Basic Microbiology 48, no. 5 (October 2008): 430–35. http://dx.doi.org/10.1002/jobm.200800008.

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8

Lynn, Elizabeth C., and Robert B. Beckstead. "Identification of Gene Expression Elements in Histomonas meleagridis Using Splinkerette PCR, a Variation of Ligated Adaptor PCR." Journal of Parasitology 98, no. 1 (February 2012): 135–41. http://dx.doi.org/10.1645/ge-2916.1.

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9

See Too, W. C., and L. L. Few. "Cloning of triose phosphate isomerase gene from an antarctic psychrophilic Pseudomonas sp. by degenerate and splinkerette PCR." World Journal of Microbiology and Biotechnology 26, no. 7 (January 3, 2010): 1251–59. http://dx.doi.org/10.1007/s11274-009-0295-9.

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10

Cleveland, Susan M., and Utpal P. Dave. "Insertional Activation of GLI2 in Adult T-Cell Leukemia/Lymphoma." Blood 110, no. 11 (November 16, 2007): 4149. http://dx.doi.org/10.1182/blood.v110.11.4149.4149.

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Abstract Retroviruses induce cancer by integrating into the cellular genome and activating oncogenes or inactivating tumor suppressor genes. Human T-cell Leukemia Virus type 1 (HTLV-1), a complex retrovirus, induces Adult T-cell Leukemia/Lymphoma (ATLL) after a latency of over 30 years and in only 5% of carriers. The long latency and incomplete penetrance is similar to how slow transforming retroviruses induce cancer in mice and imply multiple oncogenic “hits” need to accumulate for clinically apparent disease. Insertional mutagenesis may be one mechanism by which ATLL develops. We used splinkerette-PCR to clone and map insertion sites from an HTLV-1 infected T-cell line, Hut-102. We identified an HTLV-1 insertion 5′ of the GLI2 gene, formerly known as Tax-Helper-Protein-1. We found GLI2 was up-regulated by promoter insertion. Interestingly, we found GLI2 protein occupied the HTLV-1 Long Terminal Repeat. The effect of GLI2 expression on viral expression was investigated by knockdown of GLI2 in Hut-102 cells. Our results show that retroviral insertional mutagenesis can be an important mechanism in HTLV-1-induced leukemias and lymphomas.
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11

Fili, A. E., A. P. Alessio, W. Garrels, D. O. Forcato, M. F. Olmos Nicotra, A. C. Liaudat, R. J. Bevacqua, et al. "242 HIGHLY EFFICIENT SLEEPING BEAUTY TRANSPOSON-MEDIATED TRANSGENESIS IN BOVINE FETAL FIBROBLASTS." Reproduction, Fertility and Development 28, no. 2 (2016): 253. http://dx.doi.org/10.1071/rdv28n2ab242.

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Active transposon-mediated transgenesis is an emerging tool for basic and applied research in livestock. We have demonstrated the effectiveness of a helper-independent piggyBac transposon (pGENIE-3) for gene transfer into the genome of bovine cells (Alessio et al. 2014 Reprod. Domest. Anim. 49, 8). Here, we extend our previous research by examining the suitability of a Sleeping Beauty (SB) transposon-based methodology to deliver transgenes into the genome of bovine fetal fibroblasts (BFF), and the ability of these cells to support in vitro embryo development upon somatic cell nuclear transfer (SCNT). In a first experiment, BFF were chemically cotransfected (JetPRIME®, Polyplus-transfection, Illkirch, France) with a helper plasmid (pCMV-SB100X), which carries an expression cassette for the SB transposase, and the donor vector (pT2/Venus/RMCE) harboring an expression cassette for a fluorescent protein (Venus) flanked by the SB inverted terminal repeats (ITR). Three different ratios of helper and donor plasmids were studied: 1 : 2, 1 : 1 and 2 : 1. After 15 days of culture, the number of fluorescent colonies was counted on an inverted microscope. When vectors were used at ratios of 1 : 1 and 2 : 1, a 78-fold and 88-fold increase (P ≤ 0.05) in the number of fluorescent colonies compared with that in the no-transposase control were calculated. In a second experiment, BFF were chemically cotransfected with the helper vector pCMV-SB100X, and 2 donor transposons: pT2/Venus/RMCE and pT2/SV40-Neo. The former harbors a neo resistance cassette framed by SB ITRs. Different ratios of helper:donors (1 : 1 : 1, 2 : 1 : 1 and 2 : 0.5 : 0.5) were studied, and each ratio compared with a no-transposase control. After 15 days of antibiotic selection, the number of G418-resistant colonies was determined. Every time a functional SB transposase vector was included, the number of fluorescent and G418-resistant colonies was markedly higher compared with that in the respective control without transposase (P ≤ 0.001). Interestingly, all G418-resistant colonies expressed Venus. Molecular characterisation of genomic insertions in 6 monoclonal cell lines was performed by PCR and splinkerette PCR. PCR analysis confirmed presence of the Venus transgene in all cell lines. Splinkerette PCR results revealed at least 15 transposase-catalyzed genomic insertions of the transgene. Individual cells from a polyclonal SB transgenic fibroblast culture were used as nuclear donors to produce zona-free SCNT embryos. Of the reconstructed embryos, 33% reached blastocyst stage and about half of them expressed Venus. In conclusion, SB transposase is able to actively transpose monomeric copies of transgenes into the genome of bovine cells, which can be reprogrammed upon nuclear transfer to generate morphologically normal embryos expressing the transgene of interest.
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12

Shen, Dan, Songlei Xue, Shuheng Chan, Yatong Sang, Saisai Wang, Yali Wang, Cai Chen, Bo Gao, Ferenc Mueller, and Chengyi Song. "Enhancer Trapping and Annotation in Zebrafish Mediated with Sleeping Beauty, piggyBac and Tol2 Transposons." Genes 9, no. 12 (December 13, 2018): 630. http://dx.doi.org/10.3390/genes9120630.

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Although transposon-mediated enhancer trapping (ET) is successfully applied in diverse models, the efficiency of various transposon systems varies significantly, and little information is available regarding efficiency of enhancer trapping by various transposons in zebrafish. Most potential enhancers (Ens) still lack evidence of actual En activity. Here, we compared the differences in ET efficiency between sleeping beauty (SB), piggyBac (PB) and Tol2 transposons. Tol2 represented the highest germline transfer efficiencies at 55.56% (NF0 = 165), followed by SB (38.36%, NF0 = 151) and PB (32.65%, NF0 = 149). ET lines generated by the Tol2 transposon tended to produce offspring with a single expression pattern per line, while PB and SB tended to generate embryos with multiple expression patterns. In our tests, 10 putative Ens (En1–10) were identified by splinkerette PCR and comparative genomic analysis. Combining the GFP expression profiles and mRNA expression patterns revealed that En1 and En2 may be involved in regulation of the expression of dlx1a and dlx2a, while En6 may be involved in regulation of the expression of line TK4 transgene and rps26, and En7 may be involved in the regulation of the expression of wnt1 and wnt10b. Most identified Ens were found to be transcribed in zebrafish embryos, and their regulatory function may involve eRNAs.
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13

Sato, Masahiro, Emi Inada, Issei Saitoh, Shingo Nakamura, and Satoshi Watanabe. "In Vivo Piggybac-Based Gene Delivery towards Murine Pancreatic Parenchyma Confers Sustained Expression of Gene of Interest." International Journal of Molecular Sciences 20, no. 13 (June 26, 2019): 3116. http://dx.doi.org/10.3390/ijms20133116.

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The pancreas is a glandular organ that functions in the digestive system and endocrine system of vertebrates. The most common disorders involving the pancreas are diabetes, pancreatitis, and pancreatic cancer. In vivo gene delivery targeting the pancreas is important for preventing or curing such diseases and for exploring the biological function of genes involved in the pathogenesis of these diseases. Our previous experiments demonstrated that adult murine pancreatic cells can be efficiently transfected by exogenous plasmid DNA following intraparenchymal injection and subsequent in vivo electroporation using tweezer-type electrodes. Unfortunately, the induced gene expression was transient. Transposon-based gene delivery, such as that facilitated by piggyBac (PB), is known to confer stable integration of a gene of interest (GOI) into host chromosomes, resulting in sustained expression of the GOI. In this study, we investigated the use of the PB transposon system to achieve stable gene expression when transferred into murine pancreatic cells using the above-mentioned technique. Expression of the GOI (coding for fluorescent protein) continued for at least 1.5 months post-gene delivery. Splinkerette-PCR-based analysis revealed the presence of the consensus sequence TTAA at the junctional portion between host chromosomes and the transgenes; however, this was not observed in all samples. This plasmid-based PB transposon system enables constitutive expression of the GOI in pancreas for potential therapeutic and biological applications.
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14

Jia, Wenzhu, Zhongxia Guan, ShaSha Shi, Kuilin Xiang, Peihong Chen, Fen Tan, Numan Ullah, et al. "The Annotation of Zebrafish Enhancer Trap Lines Generated with PB Transposon." Current Issues in Molecular Biology 44, no. 6 (June 2, 2022): 2614–21. http://dx.doi.org/10.3390/cimb44060178.

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An enhancer trap (ET) mediated by a transposon is an effective method for functional gene research. Here, an ET system based on a PB transposon that carries a mini Krt4 promoter (the keratin4 minimal promoter from zebrafish) and the green fluorescent protein gene (GFP) has been used to produce zebrafish ET lines. One enhancer trap line with eye-specific expression GFP named EYE was used to identify the trapped enhancers and genes. Firstly, GFP showed a temporal and spatial expression pattern with whole-embryo expression at 6, 12, and 24 hpf stages and eye-specific expression from 2 to 7 dpf. Then, the genome insertion sites were detected by splinkerette PCR (spPCR). The Krt4-GFP was inserted into the fourth intron of the gene itgav (integrin, alpha V) in chromosome 9 of the zebrafish genome, with the GFP direction the same as that of the itgav gene. By the alignment of homologous gene sequences in different species, three predicted endogenous enhancers were obtained. The trapped endogenous gene itgav, whose overexpression is related to hepatocellular carcinoma, showed a similar expression pattern as GFP detected by in situ hybridization, which suggested that GFP and itgav were possibly regulated by the same enhancers. In short, the zebrafish enhancer trap lines generated by the PB transposon-mediated enhancer trap technology in this study were valuable resources as visual markers to study the regulators and genes. This work provides an efficient method to identify and isolate tissue-specific enhancer sequences.
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15

Salnikov, P. A., A. A. Khabarova, G. S. Koksharova, R. V. Mungalov, P. S. Belokopytova, I. E. Pristyazhnuk, A. R. Nurislamov, P. Somatich, M. M. Gridina, and V. S. Fishman. "Here and there: the double-side transgene localization." Vavilov Journal of Genetics and Breeding 25, no. 6 (October 23, 2021): 607–12. http://dx.doi.org/10.18699/vj21.068.

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Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene annotation by transposon-insertion sequencing, for identification of transcription factor binding sites and regulatory sequences, and for dissecting chromatin position effects. Precise localization of transgenes and accurate artifact filtration are essential for this type of method. To date, many mapping assays have been developed, including Inverse-PCR, TLA, LAM-PCR, and splinkerette PCR. However, none of them is able to ensure localization of both transgene’s flanking regions simultaneously, which would be necessary for some applications. Here we proposed a cheap and simple NGS-based approach that overcomes this limitation. The developed assay requires using intentionally designed vectors that lack recognition sites of one or a set of restriction enzymes used for DNA fragmentation. By looping and sequencing these DNA fragments, we obtain special data that allows us to link the two flanking regions of the transposon. This can be useful for precise insertion mapping and for screening approaches in the field of chromosome engineering, where chromosomal recombination events between transgenes occur in a cell population. To demonstrate the method’s feasibility, we applied it for mapping SB transposon integration in the human HAP1 cell line. Our technique allowed us to efficiently localize genomic transposon integrations, which was confirmed via PCR analysis. For practical application of this approach, we proposed a set of recommendations and a normalization strategy. The developed method can be used for multiplex transgene localization and detection of rearrangements between them.
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16

Hasemann, Marie S., Annette B. Sørensen, Finn S. Pedersen, Claus Nerlov, and Bo Porse. "Retroviral Insertional Mutagenesis Screen in a C/EBPalpha Proliferative Genetic Background." Blood 108, no. 11 (November 16, 2006): 4342. http://dx.doi.org/10.1182/blood.v108.11.4342.4342.

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Abstract 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. Consistently, mice lacking C/EBPalpha have no mature neutrophils and die within a few hours after birth. In contrast, homozygous knockin mice in which the wild type Cebpa allele has been replaced with a mutant allele (BRM2) deficient in repressing the activity of E2F family members, are viable. At 8 weeks of age these animals display myeloid dysplasia with absence of neutrophil granulocytes. Strikingly, in older BRM2/BRM2 knockin mice the myeloid dysplastic phenotype progress into other myeloid malignancies such as myeloid proliferative syndrome and acute myeloid leukemia. These findings strongly suggest that secondary mutations in other loci must occur during the phenotypic progression. In order to identify genes that cooperate with C/EBPalpha in the development of leukemia in BRM2/BRM2 mice a so-called retroviral insertion mutagenesis screen was performed. Inbred newborn BRM2/BRM2 and wildtype mice were injected with SRS19-6 retrovirus and when disease is evident the mice are euthanized and analyzed. As expected the BRM2/BRM2 mice have a shorter latency than wildtype mice (182 vs. 260 days). The mice have enlarged spleen, thymus, and lymph nodes and were further characterized by histology, flow cytometry and Southern blotting in order to determine the hematopoietic phenotypes. Most abundantly was the AML-like phenotype, but also T-cell lymphomas are developing. Finally, the loci carrying retroviral insertions loci are identified through a splinkerette-aided PCR strategy. This study provides a better understanding of the genes involved in the development of myeloid leukemia.
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17

Qureshi, Safia J., David J. Porteous, and Anthony J. Brookes. "Alu-based vectorettes and splinkerettes." Genetic Analysis: Biomolecular Engineering 11, no. 4 (January 1994): 95–101. http://dx.doi.org/10.1016/1050-3862(94)90046-9.

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18

Li, Zhe, Jan-Henning Klusmann, Frank J. Godinho, Hee-Won Lee, Dirk Reinhardt, and Stuart H. Orkin. "A Genome-Wide Retroviral Insertional Mutagenesis Screen for Genes Cooperating with Truncated, Oncogenic GATA1s." Blood 106, no. 11 (November 16, 2005): 2990. http://dx.doi.org/10.1182/blood.v106.11.2990.2990.

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Abstract Somatic mutations in the hematopoietic transcription factor GATA1 are found in megakaryoblasts of Down Syndrome (DS) patients with transient myeloproliferative disorder (TMD, or transient leukemia or TL) and the related acute megakaryoblastic leukemia (DS-AMKL, or DS- AML M7). These mutations lead to production of a GATA1 variant (GATA1s) lacking its N-terminal domain. Mice carrying GATA1s mutation have normal adult hematopoiesis. However, during embryonic/fetal development, we have identified a transient population of abnormal yolk sac/fetal liver megakaryocytic progenitors in mutant mice. We proposed that these progenitor cells are the target for transformation in DS-AMKL/TMD. GATA1s mice (either during development or as adults) do not develop myeloproliferative disorder or leukemia. To recapitulate human DS TMD in mice, we bred GATA1s mice to mouse DS models (Ts65Dn and Ts1Cje) and generated GATA1s/DS double mutants. The phenotype of GATA1s/DS mice is not different from that of GATA1s mice, suggesting that these mouse DS models (representing ~166 and 112 trisomic genes on human chromosome 21, respectively, including Runx1, Ets2, and Erg) do not accurately recapitulate the effects of trisomy in DS. To search for genes that cooperate with GATA1s in an unbiased fashion, we established a genome-wide retroviral insertional mutagenesis screen. GATA1s mutant fetal liver progenitors proliferate in culture in the presence of thrombopoietin (Tpo) for about 4–5 weeks. We infected mutant fetal progenitors with MSCV retrovirus and selected in vitro in the presence of Tpo for immortalized cell lines. Retroviral integration sites in these cell lines were determined by Splinkerette PCR, and confirmed by genomic PCR. Genes that were affected by retroviral integration were confirmed by real-time PCR for their elevated expression or knock-down. From the genetic screen performed thus far, we identified two common retroviral integration sites, Evi1 and Prdm16 (PR domain containing 16). Interestingly, Evi1 is also overexpressed in M7 leukemias, though its expression in non-DS M7 leukemia is higher than in DS M7 leukemia. By retroviral overexpression, we have confirmed that ectopic expression of Evi1 in GATA1s mutant fetal progenitors further enhanced proliferation. Currently we are testing the in vivo leukemogenic abilities of these cell lines by transplantation. By this approach, we will identify genes that cooperate with GATA1s in cellular transformation and, thereby, gain insights into the mechanism of leukemogenesis in DS-AMKL/TMD.
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19

Devon, Rebecca S., David J. Porteous, and Anthony J. Brookes. "Splinkerettes—improved vectorettes for greater efficiency in PCR walking." Nucleic Acids Research 23, no. 9 (1995): 1644–45. http://dx.doi.org/10.1093/nar/23.9.1644.

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20

Tolar, Jakub, Mark Osborn, Scott Bell, Lily Xia, Megan Riddle, Angela Panoskaltsis-Mortari, Scott McIvor, et al. "Transgenesis of Multipotent Adult Progenitor Cells (MAPC) with Sleeping Beauty Transposons to Determine MAPC Homing and Persistence in Real-Time In Vivo." Blood 104, no. 11 (November 16, 2004): 2099. http://dx.doi.org/10.1182/blood.v104.11.2099.2099.

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Abstract MAPC are non-hematopoietic stem cells with the capacity to form most, if not all, cell types of the body. To date, the observations of homing of the MAPC have been limited to post mortem analyses. As MAPC may be useful in cellular therapies, our goal was to map their biodistribution in live organisms. To determine the real-time organ-specific homing pattern of donor MAPC, MAPC (from BM of C57BL/6J-rosa26 mice) were co-nucleofected with cDNAs encoding the red fluorescent protein DsRed2 and luciferase, using the Sleeping Beauty (SB) transposon system. Non-viral gene transfer mediated by SB is potentially advantageous to viral gene transfer because transposons may be less immunogenic since no viral proteins are present, and they are relatively easy to produce. DsRed2 and luciferase genes were cloned into plasmid vectors containing the transposase recognition sequences flanking the reporter genes (pT/CAGGS-DsRed2; pT/CAGGS-Luciferase). MAPC (106) were co-nucleofected (Amaxa, setting T-20, buffer T) with 5mcg of each marker plasmid and the SB transposase plasmid (p/CMV-HSB2) at a 1:50 ratio. 19% of MAPC expressed DsRed2 7 days after nucleofection. The MAPC were FACS sorted (1 cell per well) for cells with the highest DsRed2 expression. All MAPC tested expressed both DsRed2 and luciferase, suggesting that co-nucleofection is an efficient means of delivery of two plasmids. Two transgenic MAPC clones selected for further analysis were confirmed to be euploid by cytogenetic analysis, and maintained differentiation potential into the three germ layers. To verify transgene integration by transposition, the genomic sites of transposon integration were determined using splinkerette PCR. In the genome of MAPC clone 1, DsRed2 transposed in two sites on chromosome 5. One integration site (5qA3) was in the 3′ untranslated region of activin receptor interacting protein 1 (Acvrinp1). In clone 2 DsRed2 transposed into a single site on chromosome 10, in an intron of a gene termed SHPRH, which encodes a putative protein with SNF2/helicase and PHD-finger domains. To investigate the real time kinetics of MAPC population after infusion, 5 x 106 DsRed2 and luciferase positive MAPC (clone 2) were infused via tail vein into 8-week-old Rag2/IL-2Rgc−/− mice (T-, B- and NK-immunodeficient mice were used as a recipient to minimize the likelihood that the host would reject donor MAPC). Using whole body imaging (Xenogen) we were able to follow the distribution of the luciferase-marked MAPC over a period of 10 weeks. In addition, using DsRed2 expression the donor MAPC-derived cells in whole lung and in lung cryosections were identified. In summary, we show for the first time stable gene expression in adult stem cells using Sleeping Beauty transposon mediated non-viral gene transfer. These results show that MAPC-based cellular therapies can be monitored in vivo and suggest that transposon-based technology may be an attractive alternative to viral based gene delivery and therapy.
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21

Stoddart, Angela, Anthony Fernald, Rachel Joy Bergerson, Jianghong Wang, Megan E. McNerney, Theodore Karrison, John Anastasi, et al. "Retroviral Insertional Mutagenesis In Egr1+/- mice, Haploinsufficient For a Human Del(5q) Myeloid Leukemia Gene, Develop Myeloid Neoplasms With Proviral Insertions In Genes Syntenic To Human 5q." Blood 122, no. 21 (November 15, 2013): 1275. http://dx.doi.org/10.1182/blood.v122.21.1275.1275.

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Abstract Therapy-related myeloid neoplasm (t-MN) is a distinctive clinical syndrome occurring after treatment with chemotherapy and/or radiotherapy, typically for a primary malignant disease. Loss of the long arm of chromosome 5, del(5q), is the most common recurring cytogenetic abnormality and is observed in 40% of t-MN patients, as well as 10-15% of patients with primary MDS or AML de novo. These deletions typically encompass over 70Mb [spanning 5q14-q33] and numerous genes, making the identification of relevant del(5q) genes very challenging. In previous studies, we identified a commonly deleted region within 5q and identified Egr1 as a del(5q) haploinsufficient myeloid leukemia gene. Using Egr1+/- mice, we previously showed that Egr1 cooperates with mutations, induced by the alkylating agent, ENU, to induce a myeloproliferative disorder with ineffective erythropoiesis (MPD). However, loss of Egr1 on its own was not sufficient for the development of MPD. To identify cooperating mutations in MPDs in Egr1 haploinsufficient mice, we conducted a retroviral insertional mutagenesis (RIM) screen. Egr1 WT (n=61) and Egr1+/- (n=77) neonates were injected with the MOL4070LTR retrovirus. Although the overall survival of MOL4070LTR-treated Egr1+/- and WT controls was similar, Egr1+/- mice developed MPD or AML with a shorter latency and at a higher overall frequency than WT littermate controls. Forty-six percent of WT mice developed myeloid disease versus 61% for Egr1+/- mice, with a median survival of 474 d for Egr1 WT and 389 d for Egr1+/- mice (p=0.03). We mapped the retroviral integration sites in myeloid neoplasms from 29 WT and 46 Egr1+/- mice using barcoded splinkerette PCR and Illumina high-throughput sequencing. To identify and analyze the statistically significant common insertion sites (CISs) we used the TAPDANCE software developed by A. Sarver. In total, 159 CISs were identified in WT mice and 365 CISs were identified in Egr1+/- mice. Several of these CIS-associated genes, such as Sox4, Pim1 and Myb have been previously identified in other genetic screens according to the Retroviral Tagged Cancer Gene Database (RTCGD). As the main goal of this study is to identify mutations that cooperate with Egr1 haploinsufficiency, we were particularly interested in CISs that were identified exclusively or more frequently in Egr1+/- mice with myeloid neoplasms. The TAPDANCE software automatically identifies associations between phenotypes and CISs using Fisher’s exact test with multiple testing correction. Using this analysis, we identified six CISs that were statistically associated with myeloid neoplasms in Egr1+/- mice, but not WT mice. The candidate cancer genes proximal to these proviral insertions included Evi1, Gfi1, Evi5, and Cd47. Analysis of transcript levels revealed elevated expression of Evi1, but not Gfi1, Evi5, or Cd47 in myeloid leukemias with proviral insertions proximal to these genes. Of interest was a CIS associated with Egr1+/- mice that mapped to a region of mouse chromosome 18 that is syntenic to human 5q31.2, proximal to the Dnajc18, Ecscr, Tmem173, Cxxc5 and Psd2 genes and adjacent to the commonly deleted region. Moreover, an analysis of co-occurring CISs revealed that this CIS co-occurred with a CIS that mapped to a region of mouse chromosome 13 that is syntenic to human 5q31.1, also deleted in t-MN patients and proximal to the Tifab, and H2afy genes. Of the genes in these two CISs, CXXC5, TMEM173, TIFAB, and H2AFY each show significantly decreased expression in bone marrow cells from t-MN del(5q) patients, consistent with haploinsufficiency. Identification of the relevant del(5q) genes in t-MN continues to be a challenge for developing therapeutic targets. Loss of expression of the tumor suppressor gene, EGR1, which is expressed at haploinsufficient levels in t-MN patients with a del(5q), on its own is not sufficient for the development of myeloid leukemia. Here we performed a forward genetic screen with Egr1+/- mice and have identified several candidate del(5q) genes, including CXXC5, TMEM173, TIFAB, and H2AFY, that should now be evaluated as candidate genes that cooperate with EGR1 haploinsufficiency in the pathogenesis of t-MN. The identification of aberrant pathways resulting from haploinsufficiency of EGR1 in cooperation with these del(5q) genes may potentially lead to the new therapeutic targets for t-MNs with chromosome 5 abnormalities. Disclosures: Largaespada: Discovery Genomics, Inc: Consultancy, Share Holder Other; NeoClone Biotechnology, Inc: Consultancy, Share Holder, Share Holder Other.
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22

Shao, Hongguang, and James Lok. "Detection of piggyBac-mediated Transposition by Splinkerette PCR in Transgenic Lines of Strongyloides ratti." BIO-PROTOCOL 4, no. 1 (2014). http://dx.doi.org/10.21769/bioprotoc.1015.

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