The transformation from ductal carcinoma in situ (DCIS) to invasive breast cancer (IBC) is a crucial step in breast cancer progression. The specific alterations that govern this transition have not been elucidated. HER2/neu is frequently overexpressed in DCIS but is less common in IBC suggesting additional requirements for transformation. To identify genes capable of cooperating with HER2/neu to fully transform mammary epithelial cells, we used an insertional mutagenesis screen on cells isolated from wild-type neu expressing mice and identified the E3 ligase HACE1 as a HER2 cooperative tumor suppressor gene. Loss of HACE1 expression is commonly seen in clinical multiple cancer datasets including breast cancer. HACE1 down-regulation in normal human mammary epithelial cells (HMECs) results in the accumulation of the activated GTP-bound Rac1 partially transforming these cells. Overexpression of HER2 activates Rac1, which further accumulates upon HACE1 loss resulting in Rac1 hyperactivation. While the
Define insertional mutagenesis. insertional mutagenesis synonyms, insertional mutagenesis pronunciation, insertional mutagenesis translation, English dictionary definition of insertional mutagenesis. Noun 1. insertional mutagenesis - a mutation caused by the insertion of exogenous DNA into a genome genetic science, genetics - the branch of biology that...
TY - JOUR. T1 - Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes. T2 - tcf7 and synembryn-like. AU - Nagayoshi, Saori. AU - Hayashi, Eriko. AU - Abe, Gembu. AU - Osato, Naoki. AU - Asakawa, Kazuhide. AU - Urasaki, Akihiro. AU - Horikawa, Kazuki. AU - Ikeo, Kazuho. AU - Takeda, Hiroyuki. AU - Kawakami, Koichi. PY - 2008/1. Y1 - 2008/1. N2 - Gene trap and enhancer trap methods using transposon or retrovirus have been recently described in zebrafish. However, insertional mutants using these methods have not been reported. We report here development of an enhancer trap method by using the Tol2 transposable element and identification and characterization of insertional mutants. We created 73 fish lines that carried single copy insertions of an enhancer trap construct, which contained the zebrafish hsp70 promoter and the GFP gene, in their genome and expressed GFP in specific cells, tissues and organs, indicating that the ...
Fast MultiSite Mutagenesis System,Mutagenesis System,Cloning and Mutagenesis System,Products,Beijing TransGen Biotech Co.Ltd,OverviewContents& storageCitations & referencesRelated ImagesDownloadOverviewDescriptionFast MultiSite Mutagenesis Syst
TY - JOUR. T1 - Cc.snf5, a gene encoding a putative component of the SWI/SNF chromatin remodeling complex, is essential for sexual development in the agaricomycete Coprinopsis cinerea. AU - Ando, Yuki. AU - Nakazawa, Takehito. AU - Oka, Kunihiko. AU - Nakahori, Kiyoshi. AU - Kamada, Takashi. PY - 2013/1. Y1 - 2013/1. N2 - We characterized a Coprinopsis cinerea mutant strain, Spe20, defective in fruiting initiation, which was isolated after restriction enzyme-mediated integration (REMI) mutagenesis of a homokaryotic fruiting strain, 326. A plasmid rescue followed by complementation experiments, RACE, and cDNA analyses revealed that the gene, a mutation of which is responsible for the phenotype, is predicted to encode a protein that exhibits a high similarity to yeast Snf5p, a key component of the chromatin remodeling complex SWI/SNF, and named Cc.snf5. Cc.Snf5 is, however, different from Snf5p in that the former has, in addition to an Snf5 domain comprising N-terminal repeat1 (rp1) and C-terminal ...
Tn-Seq is an experimental method for probing the functions of genes through construction of complex random transposon insertion libraries and quantification of each mutants abundance using next-generation sequencing. An important emerging application of Tn-Seq is for identifying genetic interactions, which involves comparing Tn mutant libraries generated in different genetic backgrounds (e.g. wild-type strain versus knockout strain). Several analytical methods have been proposed for analyzing Tn-Seq data to identify genetic interactions, including estimating relative fitness ratios and fitting a generalized linear model. However, these have limitations which necessitate an improved approach. We present a hierarchical Bayesian method for identifying genetic interactions through quantifying the statistical significance of changes in enrichment. The analysis involves a four-way comparison of insertion counts across datasets to identify transposon mutants that differentially affect bacterial fitness
Double transgenic Xenopus laevis embryos were generated using restriction enzyme-mediated integration using SV/Venus/cSCL+19 and gamma-crystallin-mCherry, which was derived from gamma-crystallin-green fluorescent protein (GFP). SV/venus/cSCL+19 enhancer (from Rita Ferreira) and cry-mCherry were co-injected ...
Insertional mutagenesis approaches use oncoretroviruses or transposons to trigger cancer in mice by widespread integration into the cellular genome and activation of oncogenes near the integration site. Mapping the genomic integration sites in tumors allows the identification of genomic regions that are recurrently hit in independent tumors (defined as Common Insertion Sites, CIS) (5), which host genes likely involved in cancer development (6).. We have recently developed a new specifically tailored LV-based insertional mutagen that allowed the induction of hepatocellular carcinoma in 3 different mouse models (4). In order to identify the integration sites from LV-induced tumors and control nontumoral samples, we exploited LAM-PCR (1) followed by 454 pyrosequencing. The analysis of LV integrations in tumors allowed the identification of 4 new liver cancer genes. Here we describe how we applied the LAM-PCR protocol1 for the retrieval of LV integrations from the hepatocellular carcinomas induced ...
Baker, Lindsey A., Tiriac, Hervé, Clevers, Hans, Tuveson, David A. (April 2016) Modeling Pancreatic Cancer with Organoids. Trends in Cancer, 2 (4). pp. 176-190. ISSN 2405-8033 Bapiro, T. E., Frese, K. K., Courtin, A., Bramhall, J. L., Madhu, B., Cook, N., Neesse, A., Griffiths, J. R., Tuveson, D. A., Jodrell, D. I., Richards, F. M. (May 2014) Gemcitabine diphosphate choline is a major metabolite linked to the Kennedy pathway in pancreatic cancer models in vivo. British Journal of Cancer, 111 (2). pp. 318-325. ISSN 0007-0920 Bergerson, R. J., Collier, L. S., Sarver, A. L., Been, R. A., Lugthart, S., Diers, M. D., Zuber, J., Rappaport, A. R., Nixon, M. J., Silverstein, K. A. T., Fan, D. H., Lamblin, A. F. J., Wolff, L., Kersey, J. H., Delwel, R., Lowe, S. W., OSullivan, M. G., Kogan, S. C., Adams, D. J., Largaespada, D. A. (May 2012) An insertional mutagenesis screen identifies genes that cooperate with Mll-AF9 in a murine leukemogenesis model. Blood, 119 (19). pp. 4512-4523. ISSN 0006-4971 Boj, ...
Knockout Sudoku allows construction of whole-genome knockout collections for a wide range of microorganisms at a lower cost and increased speed, using combinatorial pooling, next-generation sequencing, and a Bayesian inference algorithm to process and annotate extremely large progenitor transposon insertion mutant collections. Knockout Sudoku is a method for the construction of whole-genome knockout collections for a wide range of microorganisms with as little as 3 weeks of dedicated labor and at a cost of ∼$10,000 for a collection for a single organism. The method uses manual 4D combinatorial pooling, next-generation sequencing, and a Bayesian inference algorithm to rapidly process and then accurately annotate the extremely large progenitor transposon insertion mutant collections needed to achieve saturating coverage of complex microbial genomes. This method is ∼100× faster and 30× lower in cost than the next comparable method (In-seq) for annotating transposon mutant collections by combinatorial
Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3 Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.. ...
Read Strategies for Mutagenesis and Gene Cloning Using Transposon Tagging and T-DNA Insertional Mutagenesis, Annual Review of Plant Biology on DeepDyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips.
The gene trap vector is introduced into ES cells by electroporation or retroviral infection and clones containing gene trap insertions are selected by antibiotic resistance. Antibiotic resistant clones are then picked into 96-well master plates, which are later replicated to generate frozen stocks for long-term storage of clones, cell lysates for RNA and DNA isolation followed by PCR-based gene identification techniques.. ...
Nusse R, van Ooyen A, Rijsewijk F, van Lohuizen M, Schuuring E, vant Veer L. Retroviral insertional mutagenesis in murine mammary cancer. Proc R Soc Lond B Biol Sci. 1985;226:3-13. Abstract ...
The IGTC focuses on creating resources of embryonic stem cells with gene trap insertions in every or most genes in the mouse genome. A brief description of the gene trap consortium is available online. The Gene Trap Consortium is also described in Nature Genetics 36: 543-544, 2004. Links to Databases of Gene Trapped ES Cell Lines:. ...
Systems and methods for isolating DNA molecules that can include the steps of: providing a transposon mutant collection, the transposon mutant collection being stored in a plurality of wells; dispatc
OFDM (orthogonal frequency division multiplexing) communication is very attractive for high data rate transmission especially in the frequency selective fa
A novel P[UAS] insertion line shows progressive behavioral defects.Crossing P[UAS]117 to the pdf-gal4 driver results in a significant decrease in the rhythmicit
Sallaud C, Gay C, Larmande P, Bès M, Piffanelli P, Piégu B, Droc G, Regad F, Bourgeois E, Meynard D et al.. 2004. High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics. The Plant journal : for cell and molecular biology. 39(3):450-64. Abstract ...
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Insertion Verification by PCR:. We will make fly DNA from transgenic stocks and perform PCR to confirm insertion at the targeted site. $25 per line.. Virgin Collection:. ...
An insertion is an addition in which an atom in one reactant is inserted between two atoms bound to each other by a covalent bond in the other reactant.. ...
Transposon insertion site profiling chip (TIP-chip) was invented by Researchers at the Johns Hopkins High Throughput Biology Center. Tip-chip can be used to help identify otherwise elusive disease-causing mutations in the 97 percent of the genome long believed to be
Promoterless gene trap vectors have been widely used for high-efficiency gene targeting and random mutagenesis in embryonic stem (ES) cells. Unfortunately, such vectors are only effective for genes expressed in ES cells and this has prompted the development of expression-independent vectors. These polyadenylation (poly A) trap vectors employ a splice donor to capture an endogenous genes polyadenylation sequence and provide transcript stability. However, the spectrum of mutations generated by these vectors appears largely restricted to the last intron of target loci due to nonsense-mediated mRNA decay (NMD) making them unsuitable for gene targeting applications. Here, we present novel poly A trap vectors that overcome the effect of NMD and also employ RNA instability sequences to improve splicing efficiency. The set of random insertions generated with these vectors show a significantly reduced insertional bias and the vectors can be targeted directly to a 5′ intron. We also show that this ...
Looking for online definition of interrupted gene in the Medical Dictionary? interrupted gene explanation free. What is interrupted gene? Meaning of interrupted gene medical term. What does interrupted gene mean?
We are attempting to identify cellular oncogenes activated in mammary tumours by using the mouse mammary tumour virus (MMTV) as an insertional mutagen. MMTV, a retrovirus lacking a host cell-derived viral oncogene, induces adenocarcinomas of the mammary gland after a long latency period. The tumours are clonal outgrowths of cells carrying one or more integrated MMTV proviral copies. We have cloned an integrated MMTV provirus with its adjacent chromosomal DNA and we have established that the insertion site was part of a domain of the mouse genome in which MMTV proviruses are inserted in many different tumours. A gene within this domain, called int-1 is transcriptionally activated as a consequence of proviral integration. We have proposed that int-1 is a cellular oncogene for mammary tumours. Proviral activation of int-1 occurs in cis, over distances of up to 10 kilobases and is presumably caused by the transcriptional enhancer present on the MMTV long terminal repeat. The putative int-1 mammary ...
The PiggyBac (PB) transposon has emerged as a novel mutagenesis tool for understanding gene function and for phenotypic screening in eukaryotes
Carcinogenesis is a multistep process involving alterations in various cellular pathways. The critical genetic events driving the evolution of primary liver cancer, specifically hepatoblastoma and hepatocellular carcinoma (HCC), are still poorly understood. However, telomere stabilization is acknowledged as prerequisite for cancer progression in humans. In this project, human fetal hepatocytes were utilized as a cell culture model for untransformed, proliferating human liver cells, with...
Azotobacter vinelandii produces the biopolymer alginate, which has a wide range of industrial and pharmaceutical applications. A random transposon insertion mutant library was constructed from A. vinelandii ATCC12518Tc in order to identify genes and pathways affecting alginate biosynthesis, and abou.... ...
Multiple methods have been introduced over the past 30 years to identify the genomic insertion sites of transposable elements and other DNA elements that integrate into genomes. However, each of these methods suffer from limitations that can frustrate attempts to map multiple insertions in a single genome and to map insertions in genomes of high complexity that contain extensive repetitive DNA. I introduce a new method for transposon mapping that is simple to perform, can accurately map multiple insertions per genome, and generates long sequence reads that facilitate mapping to complex genomes. The method, called TagMap, for Tagmentation-based Mapping, relies on a modified Tn5 tagmentation protocol with a single tagmentation adaptor followed by PCR using primers specific to the tranposable element and the adaptor sequence. Several minor modifications to normal tagmentation reagents and protocols allow easy and rapid preparation of TagMap libraries. Short read sequencing starting from the adaptor ...
Transposon mutagenesis is one of the most widely used strategies to generate a large number of random mutations within a bacterial genome and then to precisely ...
Transposons have been used in invertebrates for transgenesis and insertional mutagens in genetic screens. We tested a functional transposon called Sleeping Beauty in the one-cell mouse embryo. In this report, we describe experiments in which transposon vectors were injected into one-cell mouse embryos with mRNA expressing the SB10 transposase enzyme. Molecular evidence of transposition was obtained by cloning of insertion sites from multiple transgenic mice produced by SB10 mRNA/transposon coinjection. We also demonstrate germ-line transmission and expression from transposed elements. This technique has promise as a germ-line transgenesis method in other vertebrate species and for insertional mutagenesis in the mouse ...
The non-coding regions of tumour cell genomes harbour a considerable fraction of total DNA sequence variation, but the functional contribution of these variants to tumorigenesis is ill-defined. Among these non-coding variants, somatic insertions are among the least well characterized due to challenges with interpreting short-read DNA sequences. Here, using a combination of Chip-seq to enrich enhancer DNA and a computational approach with multiple DNA alignment procedures, we identify enhancer-associated small insertion variants. Among the 102 tumour cell genomes we analyse, small insertions are frequently observed in enhancer DNA sequences near known oncogenes. Further study of one insertion, somatically acquired in primary leukaemia tumour genomes, reveals that it nucleates formation of an active enhancer that drives expression of the LMO2 oncogene. The approach described here to identify enhancer-associated small insertion variants provides a foundation for further study of these abnormalities ...
An alternative to insulin injections is the insulin pump. The pump is a computerized device, about the size of a beeper or pager, often worn on a belt or in a pocket. The pump delivers a continuous low (basal) dose of insulin through a cannula (a flexible plastic tube), which attaches to the body through a small needle inserted into the skin. The cannula is taped in place and the needle is removed. Common insertion sites on the body include the thighs, buttocks, abdomen, upper arms and other areas with fatty tissue.. When a person wearing a pump eats, she pushes a button on the pump to deliver an extra amount of insulin called a bolus to provide insulin for their food.. The advantages of the pump include:. ...
DNA signature tags (molecular barcodes) facilitate functional screens by identifying mutants in mixed populations that have a reduced or increased adaptation to a particular environment. Many innovative adaptations and refinements in the technology have been described since its original use with Salmonella; they have yielded a wealth of information on a broad range of biological processes--mainly in bacteria, but also in yeast and other fungi, viruses, parasites and, most recently, in mammalian cells. By combining whole-genome microarrays and comprehensive ordered libraries of mutants, high-throughput functional screens can now be achieved on a genomic scale.
Despite the recent completion of the human genome project, an ostensibly more difficult post-genomic challenge will be the functional annotation of all human genes and integration of this information into an operational cell-based model. Unfortunately, ...
An ampicillin resistance plasmid carrying the cloned repressor gene cII of the L phage (Salmonella lyphimurium) was conducted by Flac into an F- recipient. Two types of plaamids were isolated from Apr transconjugants. The majority of plasmids were dimers with one copy of Tn1000 inserted, the minority being monomers with one copy of Tn1000. This proportion remained unaltered when we used the Flac strain transformed with a monomeric form of the recombinant plasmid as a donor. An extensive oligomerization of pBR322-originating plasmids was proved in the presence of Flac; its presumable relationship to transposition-related processes is suggested.
Transposon insertion in ykyB increases the activation of an artificial ComK feedback loop.Strains PG401 (amyE::PcomG-lacZ-gfp, PcomG-comK, ΔmecA) and PG401-Tn4
An important aspect of functional genomics is understanding the phenotypic consequence of mutations in all genes in a genome. A comprehensive collection of gene knockouts allows a defined set of mutations to be systematically studied for more efficient association of genes to functions (reviewed in [1]). Multiple approaches have been used to develop comprehensive knockout resources. Biological differences between organisms make specific technologies such as homologous recombination, RNA interference, or insertional mutagenesis more useful in generating a resource for an individual species. In plants, a comprehensive knockout collection was generated for Arabidopsis thaliana via mutagenesis with insertion tags [2-5]. Genomic DNA flanking each tag was systematically amplified and sequenced from each mutant. These Flanking Sequence Tags (FSTs) index each mutant to the genome and are accessible through the SIGnAL T-DNA Express database, which links the mutant stocks to genome annotations [6]. A ...
Zebrafish is an excellent model animal to study vertebrate development by genetic approaches. Hundreds of mutations affecting various processes of development have been isolated by chemical mutagenesis and insertional mutagenesis using a pseudotyped retrovirus. However, useful transposon tools and m …
에탄올, 고온, 저해제 스트레스 상황에서 내성을 가지는 사카로마이세스 세레비지아에 균주는 에탄올 생산 산업을 위해 중요한 부분을 차지한다. 본 연구에서는 에탄올 15 % 까지 내성을 가지는 5개의 균주를 트랜스포존에 의해 돌연변이가 일어난 집단에서 테스트를 통해 분리했다. 5개중에 2개는 고온 (42 °C)에도 내성을 가지는 것을 조사되었고, 그 중에서 1개는 푸르푸랄 50 mM까지 내성을 가지는 것을 확인했다. 트랜스포존이 끼워 들어간 위치를 통해 7개의 추정 유전자 (CMP2, IMD4, SSK2, PPG1, DLD3, PAM1, MSN2)를 확인하고, 노던 블럿 분석법을 통해 CMP2와 IMD4, SSK2와 PPG1유전자가 동시에 하향조절 되었고, DLD3 유전자또한 하향조절, PAM1과 MSN2의 전사 해독 틀이 깨져있는 것을 확인 할 수 있었다. 이 7 유전자가 독립적으로 망가진 돌연변이는 에탄올 내성이 있었고, 그들 ...
These OVE#2489A mice harbor a mutation created by random insertion of the SB-sa-IRES-rtTA-pA-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2223). The donating investigator reports the phenotype of homozygous mice as: embryonic day (E)8 lethal.
These OVE#2524B mice harbor a mutation created by random insertion of the SB-cHS4core-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2229). The donating investigator reports the phenotype of homozygous mice as: renal agenesis.
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TY - JOUR. T1 - Insertional mutagenesis and cloning of the genes required for biosynthesis of the host-specific AK-toxin in the Japanese pear pathotype of Alternaria alternata. AU - Tanaka, Aiko. AU - Shiotani, Hiroshi. AU - Yamamoto, Mikihiro. AU - Tsuge, Takashi. N1 - Copyright: Copyright 2017 Elsevier B.V., All rights reserved.. PY - 1999/8. Y1 - 1999/8. N2 - The Japanese pear pathotype of Alternaria alternata causes black spot of Japanese pear by producing a host-specific toxin known as AK-toxin. Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for toxin biosynthesis. Protoplasts of a wild-type strain were treated with a linearized plasmid along with the restriction enzyme used to linearize the plasmid. Of 984 REMI transformants recovered, three produced no detectable AK-toxin and lost pathogenicity on pear leaves. Genomic DNA flanking the integrated plasmid was recovered from one of the mutants. With the recovered DNA used as a probe, a cosmid clone ...
Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The Piggybac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 20 mouse lines to be compatible with both transposases in constitutive, tissue-, or temporal-specific mutagenesis. Mice with ...
We report an SB insertional mutagenesis screen to identify loci that cooperate with oncogenic Kras to drive pancreatic adenocarcinoma in the mouse. SB-driven pancreatic adenocarcinomas show all stages of mPanIN that develop into early noninvasive adenocarcinoma and finally, invasive, highly metastatic adenocarcinoma. Importantly, these tumors also have a high desmoplastic component, a predominant histopathic feature of human pancreatic cancer. The T2Onc3 transposon proved to be more potent than T2Onc2 in inducing metastatic tumors. The exact mechanism for the difference between the two transposons in driving pancreatic cancer is unknown. One possibility is that the location of the transposon concatemer donor influences the development of the tumor. The T2Onc3 donor resides on chromosome 9, and pancreatic cancer genes in close proximity may be frequently mutated in these tumors because of local hopping.. Both T2Onc2 and T2Onc3 cohorts exhibited considerably higher numbers of nonredundant ...
High-throughput analysis of genome-wide transposon mutant libraries is a powerful tool for (conditional) essential gene discovery. Recently, several next generation sequencing approaches, e.g. Tn-seq, INseq and TraDIS, have been developed that accurately map the site of transposon insertions by mutant-specific amplification and sequence readout of DNA flanking the transposon insertions site, assigning a measure of essentiality based on the number of reads per gene or per mutant. However, analysis of these large and complex datasets is hampered by the lack of an easy to use and automated tool for transposon insertion sequencing data ...
The extraintestinal pathogen, avian pathogenic E. coli (APEC), known to cause systemic infections in chickens, is responsible for large economic losses in the poultry industry worldwide. In order to identify genes involved in the early essential stages of pathogenesis, namely adhesion and colonization, Signature-tagged mutagenesis (STM) was applied to a previously established lung colonization model of infection by generating and screening a total of 1,800 mutants of an APEC strain IMT5155 (O2:K1:H5; Sequence type complex 95). The study led to the identification of new genes of interest, including two adhesins, one of which coded for a novel APEC fimbrial adhesin (Yqi) not described for its role in APEC pathogenesis to date. Its gene product has been temporarily designated ExPEC Adhesin I (EA/I) until the adhesin-specific receptor is identified. Deletion of the ExPEC adhesin I gene resulted in reduced colonization ability by APEC strain IMT5155 both in vitro and in vivo. Furthermore, complementation of
In this study, we created a fission yeast insertion mutant library in which all mutants were tagged with unique barcode sequences and stored as two readily available selection platforms. The 384-well mutant arrays allow genetic screens on individual mutants and can be extended to genetic approaches such as synthetic genetic array (SGA) [45, 46]. These mutant arrays have been used to identify mutants with four distinct phenotypes (Table 2) as well as strains that are hyper-sensitive to cancer chemotherapeutics camptothecin and bleomycin (Hale and Runge, unpublished data). In addition to 384-well mutant arrays, mutant pools of 1800 mutants are available for parallel analysis.. The insertion mutagenesis used in this study relied on random non-homologous recombination, where a vast majority of transformants have unstable, circularized vector DNA and only a small portion have stable insertions. To facilitate the collection of stable insertion mutants, we included low-dose 5-FOA in our initial ...
TY - JOUR. T1 - A Tn10 derivative (T-POP) for isolation of insertions with conditional (tetracycline-dependent) phenotypes. AU - Rappleye, Chad A.. AU - Roth, John R.. PY - 1997/9. Y1 - 1997/9. N2 - A new Tn10-based transposon has been constructed and used to isolate insertion mutations with tetracycline-conditional phenotypes. Classes of mutants include conditional lethal mutations, conditional auxotrophs, and conditional mutants of the eut (ethanolamine utilization) operon. The described mutations were made with a new derivative of Tn10dTet that we have called Tn10d(T-POP). Others have noted that transposon Tn10dTet directs weak tetracycline-inducible transcripts out of both ends of the element into adjacent sequences. We have increased this level of outward transcription from Tn10dTet by selecting deletion mutations within the element that presumably remove transcription-termination signals. Insertion of the Tn10d(T-POP) element within an operon disrupts the target gene and makes expression ...
In this communication, we report a novel strategy for the genetic manipulation of large viral DNA genomes. In a single step we cloned an infectious cytomegalovirus DNA as a bacterial artificial chromosome in E. coli and reconstituted virus progeny after transfection of the BAC plasmid into eukaryotic cells. This approach makes the CMV genome accessible to the genetic techniques established for E. coli. As an example for the power of the mutagenesis procedures, we performed a targeted insertion of four nucleotides into the 230-kb MCMV genome. In principle, any mutation (point mutations, insertions, and deletions) in any region of the genome can now be introduced using the described mutagenesis procedure. Moreover, other procedures, for example a random transposon mutagenesis of the CMV genome are conceivable. Multiple mutations can be introduced in consecutive rounds of mutagenesis without the need to reconstitute infectious viral intermediates. Construction of revertant genomes can be easily ...
Chapter 1: Introduction: Author: Anton Berns (Netherlands Cancer Institute) -- Chapter 2: Retroviral mutagenesis in mouse leukemia/lymphoma: Author: David Largaespada, Ph.D. (University of Minnesota) -- Chapter 3: MMTV models of breast cancer: Author: John Hilkens (NKI) -- Chapter 4: Retroviral mutagenesis in other organisms: Author: Michael Dvorak (Inst. Of Molecular Genetics, Prague, Czech Rep.) -- Chapter 5: Sleeping Beauty models of cancer: Author: Adam J. Dupuy, Ph.D. (University of Iowa) -- Chapter 6: Insertional mutagenesis in gene therapy patients: Author: David Williams, M.D. (Cincinnati Children?셲 Hospital) -- Chapter 7: Bioinformatics of high throughput insertional mutagenesis: Author: Keiko Akagi (NCI-Frederick ...
Active retrotransposons play important roles during evolution and continue to shape our genomes today, especially in genetic polymorphisms underlying a diverse set of diseases. However, studies of human retrotransposon insertion polymorphisms (RIPs) based on whole-genome deep sequencing at the population level have not been sufficiently undertaken, despite the obvious need for a thorough characterization of RIPs in the general population.|br| Herein, we present a novel and efficient computational tool named Specific Insertions Detector (SID) for the detection of non-reference RIPs. We demonstrate that SID is suitable for high depth whole-genome sequencing (WGS) data using paired-end reads obtained from simulated and real datasets. We construct a comprehensive RIP database using a large population of 90 Han Chinese individuals with a mean 68× depth per individual. In total, we identify 9342 recent RIPs, and 8433 of these RIPs are novel compared with dbRIP, including 5826 Alu, 2169 long interspersed
A fixed gene copy number is important for the in silico construction of engineered synthetic networks. However, the copy number of integrated genes depends on their genomic location. This gene dosage effect is rarely addressed in synthetic biology. Two studies in Escherichia colipresented conflicting data on the impact of gene dosage. Here, we investigate how genome location and gene orientation influences expression in Bacillus subtilis. An important difference with the E. coli studies is that we used an unbiased genome integration approach mediated by random transposon insertion. We found that there is a strong gene dosage effect in fast growing B. subtilis cells, which can amount to a 5-fold difference in gene expression. In contrast, gene orientation with respect to DNA replication direction does not influence gene expression. Our study shows that gene dosage should be taken into account when designing synthetic circuits in B. subtilis and presumably other bacteria.. ...
Institutions: Samuel Lunenfeld Research Institute We are performing gene trap-based expression and genotypic screens to generate new mouse mutations that will help delineate the molecular controls of specific developmental programs. Using a polyA trap vector with recombination sites for post-insertional manipulations, gene trap insertions are screened using multiplexed in vitro differentiation and induction assays. An expression profile is being generated at a rate of greater than 5000 per year. Sequence tags for all insertions demonstrating restricted expression patterns (about 20%) are now being generated. A database is being developed that will be searchable by expression pattern, sequence, and phenotype. The clones will be available as a resource to researchers worldwide. One of the clones identified in our ongoing screen, SNAG-1, is expressed by hematopoietic stem cells, neural endothelial cells, cardiomyocytes and sensory nerves. SNAG-1 mutant embryos die mid-gestation with neural vascular ...
The mini-Tn5 are mobile elements able to transpose to the chromosome from a delivery plasmid, by means of the transposase activity encoded by the tnp* gene, which is present in cis in the plasmid but external to the mini-Tn5 element. pUT mini-Tn5 vectors can be used to insert any DNA fragment in the bacterial chromosome, by cloning it within the mini-Tn5 region. Due to the loss of the tnp* gene after insertion, minitransposons are stably integrated into the chromosome and inherited. The different pUTmini-Tn5 vectors can then be used for repeated insertion events and therefore multiple insertions in the same strain are only limited by the availability of distinct selection markers.. Advantages:. ...
The Genetics Society of America (GSA), founded in 1931, is the professional membership organization for scientific researchers and educators in the field of genetics. Our members work to advance knowledge in the basic mechanisms of inheritance, from the molecular to the population level.. Online ISSN: 1943-2631. ...
Atarés Huerta, Alejandro; Moyano, Elena; Morales, Belén; Schleicher, Peter; García Abellán, José Osvaldo; Antón Martínez, María Teresa; García Sogo, Begoña; Pérez Martin, Fernando; Lozano, Rafael; Borja Flores, Francisco; Moreno Ferrero, Vicente; Bolarin Jimenez, Maria del Carmen; Pineda Chaza, Benito José (Springer Verlag (Germany), 2011) ...
A new paper in Cell Reports utilizes RNA-seq and Tn-seq (the tn in tn-seq stands for transposon) to map the transcriptional and fitness changes in bacterial gene networks in response to stressors, like nutrient depletion and antibiotics.. The transcriptional response measures changes in gene expression as measured by RNA-seq. The fitness or phenotypic response describes the importance of each gene to the response. This is measured by a different assay, Tn-seq, which takes advantage of transposon insertion to selectively inactivate genes in the bacterial genome. Those genes that are depleted in the stressor condition are determined to be high fitness (owing to the fact that the bacteria without those genes died under stress).. First, before even considering DE genes, they found that there is no correlation between a genes transcriptional abundance (not fold change) and its fitness. While most high-fitness genes were also high abundance, many more high-abundance genes were not high-fitness. ...
From the nomenclature guidelines: Newly generated Transposon insertions, especially those located in apparently intergenic regions, may also be given Ti (transposon insertion) names. These consist of the designation identifying the laboratory of origin, the two letters Ti, and a number, all italicized. Example: eTi13 is an insertion of a Mos transposon into an intergenic region on LGIII ...
A DNA transposon, or jumping gene, controls its amplification within a genome through a competition between the enzyme multimers that are responsible for its mobility.
Interestingly there is no rule such as the higher evolved an organism the more transposable elements. Although we find more transposable elements in higher evolved organisms this rule can not be maintained if compared single species as frogs and humans.. Transposons most often code for transposase the enzyme responsible for the transposon dislocation. As Transposons are so common in a genome it is not surprising that transposases are the most common genes in a genome [2].. ...
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The Rosa26 gene locus is well suited for gene over-expression. Reduced development time and cost with ready-to-use targeting vector.
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It is possible that there is an internal rearrangement of the MiMIC transposon involving the 3prime end. May be segregating SM6a ...