Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis

ABSTRACTTransgenesis has been a mainstay of mouse genetics for over 30 years, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported due in part to limitations in the discovery tools. Targeted Locus Amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events, and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.

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Rapid evolution at the Drosophila telomere: transposable element dynamics at an intrinsically unstable locus

Genetics ◽

10.1093/genetics/iyaa027 ◽

2020 ◽

Vol 217 (2) ◽

Author(s):

Michael P McGurk ◽

Anne-Marie Dion-Côté ◽

Daniel A Barbash

Keyword(s):

Copy Number ◽

Large Scale ◽

Insertion Site ◽

Rapid Evolution ◽

Interspecific Variation ◽

High Rate ◽

Evolutionary Strategy ◽

Control Mechanisms ◽

Genetic Conflict ◽

Number Variation

AbstractDrosophila telomeres have been maintained by three families of active transposable elements (TEs), HeT-A, TAHRE, and TART, collectively referred to as HTTs, for tens of millions of years, which contrasts with an unusually high degree of HTT interspecific variation. While the impacts of conflict and domestication are often invoked to explain HTT variation, the telomeres are unstable structures such that neutral mutational processes and evolutionary tradeoffs may also drive HTT evolution. We leveraged population genomic data to analyze nearly 10,000 HTT insertions in 85 Drosophila melanogaster genomes and compared their variation to other more typical TE families. We observe that occasional large-scale copy number expansions of both HTTs and other TE families occur, highlighting that the HTTs are, like their feral cousins, typically repressed but primed to take over given the opportunity. However, large expansions of HTTs are not caused by the runaway activity of any particular HTT subfamilies or even associated with telomere-specific TE activity, as might be expected if HTTs are in strong genetic conflict with their hosts. Rather than conflict, we instead suggest that distinctive aspects of HTT copy number variation and sequence diversity largely reflect telomere instability, with HTT insertions being lost at much higher rates than other TEs elsewhere in the genome. We extend previous observations that telomere deletions occur at a high rate, and surprisingly discover that more than one-third do not appear to have been healed with an HTT insertion. We also report that some HTT families may be preferentially activated by the erosion of whole telomeres, implying the existence of HTT-specific host control mechanisms. We further suggest that the persistent telomere localization of HTTs may reflect a highly successful evolutionary strategy that trades away a stable insertion site in order to have reduced impact on the host genome. We propose that HTT evolution is driven by multiple processes, with niche specialization and telomere instability being previously underappreciated and likely predominant.

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Automated Retroviral Insertion Site Sequence Analysis and Mapping Tool Followed by Database Analysis.

Blood ◽

10.1182/blood.v106.11.5528.5528 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5528-5528

Author(s):

Stephanie Laufs ◽

Frank A. Giordano ◽

Agnes Hotz-Wagenblatt ◽

Uwe Appelt ◽

Daniel Lauterborn ◽

...

Keyword(s):

Insertional Mutagenesis ◽

Integration Site ◽

Query Sequence ◽

Cpg Islands ◽

Insertion Site ◽

High Throughput Analysis ◽

Site Analysis ◽

Conventional Analysis ◽

Integration Site Analysis ◽

Insertion Sites

Abstract Increasing use of retroviral vector-mediated gene transfer and recent reports on insertional mutagenesis in mice and humans created intense interest to characterize vector integrations on the genomic level. Techniques to determine insertion sites, mainly based on time consuming manual data processing and compilation, are thus commonly applied in gene therapy laboratories. Since a high variability in processing methods hampers further data comparison, there is an urgent need to systematically process the data arising from such analysis. The obtained sequences from the integration site analysis are judged to be authentic only if the matching part of the genomic query sequence is surrounded by the 5′LTR-sequence on the one side and the adapter-sequence on the other side. Therefore we developed an Integrationseq tool. In this task, different methods for converting the ABI sequence trace files to high quality sequences and for recognizing and deleting the LTR and adaptor parts of the isolated clones were implemented. If neither a primer nor a LTR could be found, the sequence is discarded. If the LTR is found on the complementary strand, the integration sequence is reversed. The remaining sequence between primer and LTR positions are taken as the n integration sequence and written to a sequence output file. We validated the Integrationseq tool using 259 trace files originating from integration site analysis (LM-PCR). Sequences can be trimmed by IntegrationSeq, leading to an increased yield of valid integration sequence detection, which has shown to be more sensitive (100%) than conventional analysis (94.3%) and 15 times faster than conventional analysis, while the specifities are equal (both 100%). Valid integration sequences get further processed with IntegrationMap for automatic genomic mapping. IntegrationMap runs 50 times faster than conventional methods and retrieves detailed information about whether integrations are located in or close to genes, the name of the gene, the exact localization in the transcriptional units and further parameters like the distance from the transcription start site to the integration. Further information, e.g. data about CpG-Islands, LINEs or SINEs, and their distances to the integration is also displayed. Output files generated by the task were found to be 99.8% identical with results retrieved by conventional mapping with the Ensembl alignment tool. Using both tools, IntegrationSeq and IntegrationMap, a validated, fast and standardized high-throughput analysis of insertion sites can be achieved for the first time.

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The development of APE-PCR for the cloning of genomic insertion sites of DNA elements

Biologia ◽

10.2478/s11756-013-0214-2 ◽

2013 ◽

Vol 68 (4) ◽

Cited By ~ 2

Author(s):

Zhongjuan Xu ◽

Yanli Li ◽

Zhengwei Mao ◽

Bin Yin

Keyword(s):

Insertional Mutagenesis ◽

Magnetic Particles ◽

Magnetic Beads ◽

Genetic Regulation ◽

Insertion Site ◽

Primer Extension ◽

Successful Implementation ◽

Nano Scale ◽

Dna Elements ◽

Insertion Sites

AbstractInsertional mutagenesis is a productive strategy for the exploration of genetic regulation of important biological and pathological processes, such as tumorigenesis. Successful implementation of this strategy depends heavily on an efficient approach to the identification of insertion sites present in the host genome. Here, we have introduced an easy and efficient protocol, called Adenosine-ended Primer Extension Polymerase Chain Reaction (APE-PCR), which represents several advantages, including the Addition technique we previously developed, primer extension approach coupled with biotin-streptavidin based purification, introduction of nano-scale magnetic particles, and digestion of DNA with a combination of enzymes. We have demonstrated that APE-PCR is able to amplify more and larger specific proviral insertion site (PIS)-derived fragments, with a lower non-specific background produced, fewer steps and less DNA samples required, flexibility in choice of restriction enzymes applied, at a lower cost. Replacement of regular magnetic beads with nano-scale ones in the protocol can further increase its power. Moreover, even with small amount of sample DNA, PISs can be recovered and analyzed. Thus, based on the results provided from this study, we believe that APE-PCR represents an efficient method in mapping of PISs and likely, the insertion sites of other types of DNA elements as well.

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Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis

Genome Research ◽

10.1101/gr.233866.117 ◽

2019 ◽

Vol 29 (3) ◽

pp. 494-505 ◽

Cited By ~ 34

Author(s):

Leslie O. Goodwin ◽

Erik Splinter ◽

Tiffany L. Davis ◽

Rachel Urban ◽

Hao He ◽

...

Keyword(s):

Large Scale ◽

Structural Variation ◽

Insertional Mutagenesis ◽

Integration Sites

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Sequence and Insertion Sites of Murine Melanoma-Associated Retrovirus

Journal of Virology ◽

10.1128/jvi.73.11.9178-9186.1999 ◽

1999 ◽

Vol 73 (11) ◽

pp. 9178-9186 ◽

Cited By ~ 28

Author(s):

Mengfeng Li ◽

Xiaojun Huang ◽

Zhenyu Zhu ◽

Elieser Gorelik

Keyword(s):

Insertional Mutagenesis ◽

Insertion Site ◽

Biological Significance ◽

Melanoma Cells ◽

Full Length ◽

Noncoding Region ◽

Chromosome 8 ◽

Coding Region ◽

Basic Leucine Zipper ◽

Insertion Sites

ABSTRACT We previously showed that B16 melanoma cells produce ecotropic melanoma-associated retrovirus (MelARV) which encodes a melanoma-associated antigen recognized by MM2-9B6 monoclonal antibody. The biological significance of MelARV in melanoma formation remains unknown. We found that infection of normal melanocytes with MelARV resulted in malignant transformation. It is likely that MelARV emerged from the defective Emv-2 provirus, a single copy of ecotropic provirus existing in the genome of C57BL/6 mice. In the present study, we cloned and sequenced the full-length MelARV genome and its insertion sites and we completed sequencing of the Emv-2 provirus. Our data show that MelARV has a typical full-length retroviral genome with high homology (98.54%) to Emv-2, indicating a close relationship between both viruses. MelARV probably emerged as a result of recombination between Emv-2 and an endogenous nonecotropic provirus. Some observed differences in the gag and polregions of MelARV might account for the restoration of productivity and infectivity of a novel retrovirus that somatically emerged during melanoma formation. MelARV does not contain any oncogene and therefore might induce transformation by insertional mutagenesis. We sequenced two insertion sites of MelARV. The first insertion site represents the 3′ coding region of the c-maf proto-oncogene at 67.0 centimorgans (cM) on chromosome 8. The c-mafproto-oncogene encodes a basic leucine zipper protein homologous to c-fos and c-jun. Insertion of MelARV in BL6 melanoma cells resulted in the up-regulation of c-maf. It is noteworthy that the Emv-2 provirus is also inserted into a noncoding region at 61.0 cM on the same chromosome 8. The second insertion site is the 3′ noncoding region of the DNA polymerase gamma (PolG) gene on chromosome 7. The expression of PolG was not affected by the MelARV insertion. Further investigation of the biological significance of MelARV in melanoma formation is being undertaken.

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Rapid evolution at the Drosophila telomere: transposable element dynamics at an intrinsically unstable locus

10.1101/782904 ◽

2019 ◽

Cited By ~ 2

Author(s):

Michael P McGurk ◽

Anne-Marie Dion-Côté ◽

Daniel A Barbash

Keyword(s):

Copy Number ◽

Large Scale ◽

Insertion Site ◽

Rapid Evolution ◽

Interspecific Variation ◽

High Rate ◽

Evolutionary Strategy ◽

Control Mechanisms ◽

Genetic Conflict ◽

Number Variation

ABSTRACTDrosophila telomeres have been maintained by three families of active transposable elements (TEs), HeT-A, TAHRE and TART, collectively referred to as HTTs, for tens of millions of years, which contrasts with an unusually high degree of HTT interspecific variation. While the impacts of conflict and domestication are often invoked to explain HTT variation, the telomeres are unstable structures such that neutral mutational processes and evolutionary tradeoffs may also drive HTT evolution. We leveraged population genomic data to analyze nearly 10,000 HTT insertions in 85 D. melanogaster genomes and compared their variation to other more typical TE families. We observe that occasional large-scale copy number expansions of both HTTs and other TE families occur, highlighting that the HTTs are, like their feral cousins, typically repressed but primed to take over given the opportunity. However, large expansions of HTTs are not caused by the runaway activity of any particular HTT subfamilies or even associated with telomere-specific TE activity, as might be expected if HTTs are in strong genetic conflict with their hosts. Rather than conflict, we suggest instead that distinctive aspects of HTT copy number variation and sequence diversity largely reflect telomere instability, with HTT insertions being lost at much higher rates than other TEs elsewhere in the genome. We extend previous observations that telomere deletions occur at a high rate, and surprisingly discover that more than a third do not appear to have been healed with an HTT insertion. We also report that some HTT families may be preferentially activated by the erosion of whole telomeres, implying the existence of HTT-specific host control mechanisms. We further suggest that the persistent telomere localization of HTTs may reflect a highly successful evolutionary strategy that trades away a stable insertion site in order to have reduced impact on the host genome. We propose that HTT evolution is driven by multiple processes with niche specialization and telomere instability being previously underappreciated and likely predominant.

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Reduced Genotoxic Risk Using Foamy Viral Vectors for the Treatment of Canine Leukocyte Adhesion Deficiency

Blood ◽

10.1182/blood.v110.11.3744.3744 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3744-3744

Author(s):

Thomas R. Bauer ◽

Mehreen Hai ◽

Rima L. Adler ◽

James M. Allen ◽

Laura M. Tuschong ◽

...

Keyword(s):

Gene Therapy ◽

Viral Vectors ◽

Insertional Mutagenesis ◽

Leukocyte Adhesion ◽

Severe Combined Immunodeficiency ◽

Insertion Site ◽

Leukocyte Adhesion Deficiency ◽

Hematopoietic Stem ◽

Insertion Sites ◽

Immunodeficiency Disease

Abstract Gammaretroviral vectors used in recent gene therapy clinical trials have lead to several successes, such as in the treatment of X-linked severe combined immunodeficiency disease, but have also resulted in insertional activation of nearby oncogenes, leading to leukemia in four patients. We previously reported the successful treatment of four dogs with canine leukocyte adhesion deficiency (CLAD), a lethal genetic immunodeficiency disease caused by defects in the leukocyte integrin CD18, by transplanting foamy viral (FV) vector (deltaphiMscv-cCD18) - transduced, autologous CD34+ hematopoietic stem cells. To date, more than 2 years post transplant, all four dogs have maintained CD18+ leukocyte levels ranging between 5–10%, completely reversing of the CLAD phenotype, and have no clinical or laboratory evidence of hematological malignancy. To assess the potential genotoxicity of the FV gene therapy in the treatment of CLAD, we compared the insertion sites (ISs) found in the FV vector treated CLAD dogs with ISs found in CLAD dogs treated by gammaretroviral (RV) vectors (PG13/Mscv-cCD18). Insertion sites were identified by DNA sequence analysis of ligation-mediated PCR (LM-PCR) or linear amplification-mediated PCR (LAM-PCR) amplicons and subsequent comparison to the dog genome (canFam 2.0, May 2005). Insertion site analysis was performed for integrations that were in or within 50 kb of Refseq genes (using mouse/human orthologs). Analysis of the ISs revealed a reduced preference for FV vector integrations near transcription start sites compared to RV vector integrations (41% vs. 48%), fewer integrations near potential oncogenes (11% vs. 16%), and fewer integrations within genes in general (41% vs. 52%), in the FV vector treated animals compared to the RV vector treated animals. These clinically relevant data suggest that a reduced insertional mutagenesis potential exists when using FV vectors compared to RV vectors, and support the use of FV vectors in the treatment of human hematopoietic stem cell diseases such as LAD.

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CONTRIBUTION OF DNA COPY-NUMBER VARIATION (CNV) TO CANCER SUSCEPTIBILITY AND LARGE-SCALE GENOME ALTERATIONS IN OSTEOSARCOMA (OS)

Clinical & Investigative Medicine ◽

10.25011/cim.v31i4.4821 ◽

2008 ◽

Vol 31 (4) ◽

pp. 19

Author(s):

I Pasic ◽

A Shlien ◽

A Novokmet ◽

C Zhang ◽

U Tabori ◽

...

Keyword(s):

Genomic Instability ◽

Large Scale ◽

Structural Variation ◽

Age Of Onset ◽

Cancer Susceptibility ◽

Small Sample Size ◽

Snp Array ◽

Tumour Suppressor Gene ◽

Small Sample ◽

International Hapmap Project

Introduction: OS, a common Li-Fraumeni syndrome (LFS)-associated neoplasm, is a common bone malignancy of children and adolescents. Sporadic OS is also characterized by young age of onset and high genomic instability, suggesting a genetic contribution to disease. This study examined the contribution of novel DNA structural variation elements, CNVs, to OS susceptibility. Given our finding of excessive constitutional DNA CNV in LFS patients, which often coincide with cancer-related genes, we hypothesized that constitutional CNV may also provide clues about the aetiology of LFS-related sporadic neoplasms like OS. Methods: CNV in blood DNA of 26 patients with sporadic OS was compared to that of 263 normal control samples from the International HapMap project, as well as 62 local controls. Analysis was performed on DNA hybridized to Affymetrix genome-wide human SNP array 6.0 by Partek Genomic Suite. Results: There was no detectable difference in average number of CNVs, CNV length, and total structural variation (product of average CNV number and length) between individuals with OS and controls. While this data is preliminary (small sample size), it argues against the presence of constitutional genomic instability in individuals with sporadic OS. Conclusion: We found that the majority of tumours from patients with sporadic OS show CN loss at chr3q13.31, raising the possibility that chr3q13.31 may represent a “driver” region in OS aetiology. In at least one OS tumour, which displays CN loss at chr3q13.31, we demonstrate decreased expression of a known tumour suppressor gene located at chr3q13.31. We are investigating the role ofchr3q13.31 in development of OS.

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Cyberstalking Victimization Model Using Criminological Theory: A Systematic Literature Review, Taxonomies, Applications, Tools, and Validations

Electronics ◽

10.3390/electronics10141670 ◽

2021 ◽

Vol 10 (14) ◽

pp. 1670

Author(s):

Waheeb Abu-Ulbeh ◽

Maryam Altalhi ◽

Laith Abualigah ◽

Abdulwahab Ali Almazroi ◽

Putra Sumari ◽

...

Keyword(s):

Data Analysis ◽

Structural Equation ◽

Large Scale ◽

Review Paper ◽

Essential Element ◽

Routine Activities ◽

Criminological Theory ◽

Equation Modeling ◽

Future Research ◽

Proposed Model

Cyberstalking is a growing anti-social problem being transformed on a large scale and in various forms. Cyberstalking detection has become increasingly popular in recent years and has technically been investigated by many researchers. However, cyberstalking victimization, an essential part of cyberstalking, has empirically received less attention from the paper community. This paper attempts to address this gap and develop a model to understand and estimate the prevalence of cyberstalking victimization. The model of this paper is produced using routine activities and lifestyle exposure theories and includes eight hypotheses. The data of this paper is collected from the 757 respondents in Jordanian universities. This review paper utilizes a quantitative approach and uses structural equation modeling for data analysis. The results revealed a modest prevalence range is more dependent on the cyberstalking type. The results also indicated that proximity to motivated offenders, suitable targets, and digital guardians significantly influences cyberstalking victimization. The outcome from moderation hypothesis testing demonstrated that age and residence have a significant effect on cyberstalking victimization. The proposed model is an essential element for assessing cyberstalking victimization among societies, which provides a valuable understanding of the prevalence of cyberstalking victimization. This can assist the researchers and practitioners for future research in the context of cyberstalking victimization.

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High-Rate Transition Metal-based Cathode Materials for Battery-Supercapacitor Hybrid Devices

Nanoscale Advances ◽

10.1039/d1na00523e ◽

2021 ◽

Author(s):

Cong Wang ◽

Zehao Song ◽

Pei Shi ◽

Lin Lv ◽

Houzhao Wan ◽

...

Keyword(s):

Large Scale ◽

Rapid Development ◽

Electronic Devices ◽

High Rate ◽

Specific Power ◽

Energy Storage Devices ◽

Storage Devices ◽

Electrochemical Devices ◽

Hybrid Devices ◽

Scale Grid

With the rapid development of portable electronic devices, electric vehicles and large-scale grid energy storage devices, it needs to reinforce specific energy and specific power of related electrochemical devices meeting...

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