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1.  Detecting Statistically Significant Common Insertion Sites in Retroviral Insertional Mutagenesis Screens 
PLoS Computational Biology  2006;2(12):e166.
Retroviral insertional mutagenesis screens, which identify genes involved in tumor development in mice, have yielded a substantial number of retroviral integration sites, and this number is expected to grow substantially due to the introduction of high-throughput screening techniques. The data of various retroviral insertional mutagenesis screens are compiled in the publicly available Retroviral Tagged Cancer Gene Database (RTCGD). Integrally analyzing these screens for the presence of common insertion sites (CISs, i.e., regions in the genome that have been hit by viral insertions in multiple independent tumors significantly more than expected by chance) requires an approach that corrects for the increased probability of finding false CISs as the amount of available data increases. Moreover, significance estimates of CISs should be established taking into account both the noise, arising from the random nature of the insertion process, as well as the bias, stemming from preferential insertion sites present in the genome and the data retrieval methodology. We introduce a framework, the kernel convolution (KC) framework, to find CISs in a noisy and biased environment using a predefined significance level while controlling the family-wise error (FWE) (the probability of detecting false CISs). Where previous methods use one, two, or three predetermined fixed scales, our method is capable of operating at any biologically relevant scale. This creates the possibility to analyze the CISs in a scale space by varying the width of the CISs, providing new insights in the behavior of CISs across multiple scales. Our method also features the possibility of including models for background bias. Using simulated data, we evaluate the KC framework using three kernel functions, the Gaussian, triangular, and rectangular kernel function. We applied the Gaussian KC to the data from the combined set of screens in the RTCGD and found that 53% of the CISs do not reach the significance threshold in this combined setting. Still, with the FWE under control, application of our method resulted in the discovery of eight novel CISs, which each have a probability less than 5% of being false detections.
A potent method for the identification of novel cancer genes is retroviral insertional mutagenesis. Mice infected with slow transforming retroviruses develop tumors because the virus inserts randomly in their genome and mutates cancer genes. The regions in the genome that are mutated in multiple independent tumors are likely to contain genes involved in tumorigenesis. As the size of these datasets increases, conventional methods to detect these so-called common insertion sites (CISs) no longer suffice, and an approach is required that can control the error independent of the dataset size. The authors introduce a framework that uses a technique called kernel density estimation to find the regions in the genome that show a significant increase in insertion density. This method is implemented over a range of scales, allowing the data to be evaluated at any relevant scale. The authors demonstrate that the framework is capable of compensating for the inherent biases in the data, such as preference for retroviruses to insert near transcriptional start sites. By better balancing the error, they are able to show that from the 361 published CISs, 150 can be identified that have a low probability of being a false detection. In addition, they discover eight novel CISs.
PMCID: PMC1676030  PMID: 17154714
2.  Identifying Cancer Driver Genes Using Replication-Incompetent Retroviral Vectors 
Cancers  2016;8(11):99.
Identifying novel genes that drive tumor metastasis and drug resistance has significant potential to improve patient outcomes. High-throughput sequencing approaches have identified cancer genes, but distinguishing driver genes from passengers remains challenging. Insertional mutagenesis screens using replication-incompetent retroviral vectors have emerged as a powerful tool to identify cancer genes. Unlike replicating retroviruses and transposons, replication-incompetent retroviral vectors lack additional mutagenesis events that can complicate the identification of driver mutations from passenger mutations. They can also be used for almost any human cancer due to the broad tropism of the vectors. Replication-incompetent retroviral vectors have the ability to dysregulate nearby cancer genes via several mechanisms including enhancer-mediated activation of gene promoters. The integrated provirus acts as a unique molecular tag for nearby candidate driver genes which can be rapidly identified using well established methods that utilize next generation sequencing and bioinformatics programs. Recently, retroviral vector screens have been used to efficiently identify candidate driver genes in prostate, breast, liver and pancreatic cancers. Validated driver genes can be potential therapeutic targets and biomarkers. In this review, we describe the emergence of retroviral insertional mutagenesis screens using replication-incompetent retroviral vectors as a novel tool to identify cancer driver genes in different cancer types.
PMCID: PMC5126759  PMID: 27792127
insertional mutagenesis; replication-incompetent retroviral vector; gammaretroviral (γRV) vector; lentiviral (LV) vector; cancer driver genes; drug targets; biomarker
3.  Novel principles of gamma-retroviral insertional transcription activation in murine leukemia virus-induced end-stage tumors 
Retrovirology  2014;11:36.
Insertional mutagenesis screens of retrovirus-induced mouse tumors have proven valuable in human cancer research and for understanding adverse effects of retroviral-based gene therapies. In previous studies, the assignment of mouse genes to individual retroviral integration sites has been based on close proximity and expression patterns of annotated genes at target positions in the genome. We here employed next-generation RNA sequencing to map retroviral-mouse chimeric junctions genome-wide, and to identify local patterns of transcription activation in T-lymphomas induced by the murine leukemia gamma-retrovirus SL3-3. Moreover, to determine epigenetic integration preferences underlying long-range gene activation by retroviruses, the colocalization propensity with common epigenetic enhancer markers (H3K4Me1 and H3K27Ac) of 6,117 integrations derived from end-stage tumors of more than 2,000 mice was examined.
We detected several novel mechanisms of retroviral insertional mutagenesis: bidirectional activation of mouse transcripts on opposite sides of a provirus including transcription of unannotated mouse sequence; sense/antisense-type activation of genes located on opposite DNA strands; tandem-type activation of distal genes that are positioned adjacently on the same DNA strand; activation of genes that are not the direct integration targets; combination-type insertional mutagenesis, in which enhancer activation, alternative chimeric splicing and retroviral promoter insertion are induced by a single retrovirus. We also show that irrespective of the distance to transcription start sites, the far majority of retroviruses in end-stage tumors colocalize with H3K4Me1 and H3K27Ac-enriched regions in murine lymphoid tissues.
We expose novel retrovirus-induced host transcription activation patterns that reach beyond a single and nearest annotated gene target. Awareness of this previously undescribed layer of complexity may prove important for elucidation of adverse effects in retroviral-based gene therapies. We also show that wild-type gamma-retroviruses are frequently positioned at enhancers, suggesting that integration into regulatory regions is specific and also subject to positive selection for sustaining long-range gene activation in end-stage tumors. Altogether, this study should prove useful for extrapolating adverse outcomes of retroviral vector therapies, and for understanding fundamental cellular regulatory principles and retroviral biology.
PMCID: PMC4098794  PMID: 24886479
Gamma-retrovirus; Insertional mutagenesis; Oncogenesis; Deep sequencing; Chromatin immunoprecipication with sequencing (ChIP-seq); Retroviral integration sites; RNA sequencing (RNA-seq)
4.  Insertional Mutagenesis and Deep Profiling Reveals Gene Hierarchies and a Myc/p53-Dependent Bottleneck in Lymphomagenesis 
PLoS Genetics  2014;10(2):e1004167.
Retroviral insertional mutagenesis (RIM) is a powerful tool for cancer genomics that was combined in this study with deep sequencing (RIM/DS) to facilitate a comprehensive analysis of lymphoma progression. Transgenic mice expressing two potent collaborating oncogenes in the germ line (CD2-MYC, -Runx2) develop rapid onset tumours that can be accelerated and rendered polyclonal by neonatal Moloney murine leukaemia virus (MoMLV) infection. RIM/DS analysis of 28 polyclonal lymphomas identified 771 common insertion sites (CISs) defining a ‘progression network’ that encompassed a remarkably large fraction of known MoMLV target genes, with further strong indications of oncogenic selection above the background of MoMLV integration preference. Progression driven by RIM was characterised as a Darwinian process of clonal competition engaging proliferation control networks downstream of cytokine and T-cell receptor signalling. Enhancer mode activation accounted for the most efficiently selected CIS target genes, including Ccr7 as the most prominent of a set of chemokine receptors driving paracrine growth stimulation and lymphoma dissemination. Another large target gene subset including candidate tumour suppressors was disrupted by intragenic insertions. A second RIM/DS screen comparing lymphomas of wild-type and parental transgenics showed that CD2-MYC tumours are virtually dependent on activation of Runx family genes in strong preference to other potent Myc collaborating genes (Gfi1, Notch1). Ikzf1 was identified as a novel collaborating gene for Runx2 and illustrated the interface between integration preference and oncogenic selection. Lymphoma target genes for MoMLV can be classified into (a) a small set of master regulators that confer self-renewal; overcoming p53 and other failsafe pathways and (b) a large group of progression genes that control autonomous proliferation in transformed cells. These findings provide insights into retroviral biology, human cancer genetics and the safety of vector-mediated gene therapy.
Author Summary
Cancers are known to arise by a series of mutational and non-mutational (epigenetic) events but the advent of cancer genome sequencing highlights the growing challenge of separating important (driver) from irrelevant (passenger) mutations. Retroviruses that induce cancer by inserting into host DNA and thereby altering key genes are valuable tools because they act as ‘tags’ to identify the critical targets. In this study we combined retroviral tagging with next generation sequencing to achieve a comprehensive description of lymphoma development and progression in transgenic mouse model systems. Our study suggests that three events may be sufficient for lymphoma development and identifies a genetic bottleneck at a small gene set that regulates tumour cell self-renewal, including the Myc oncogene and the p53 tumour suppressor. In contrast, many genes can provide the final step where the lymphoma cell acquires the ability to divide independently of external stimuli. As many of the target genes are conserved and play roles in cancers of non-viral origin, this study may provide a paradigm for the gene interactions that underlie cancer biology. It also elucidates the risks entailed in the recent use of retrovirus-based vectors for human gene therapy.
PMCID: PMC3937229  PMID: 24586197
5.  A recessive genetic screen for host factors required for retroviral infection in a library of insertionally mutated Blm-deficient embryonic stem cells 
Genome Biology  2007;8(4):R48.
A recessive genetic screen of an insertionally mutated Blm-/- ES cell library identifies host factors required for retroviral infection, and confirms that mCat-1 is the ecotropic murine leukaemia virus receptor in ES cells.
Host factors required for retroviral infection are potential targets for the modulation of diseases caused by retroviruses. During the retroviral life cycle, numerous cellular factors interact with the virus and play an essential role in infection. Cultured embryonic stem (ES) cells are susceptible to retroviral infection, therefore providing access to all of the genes required for this process to take place. In order to identify the host factors involved in retroviral infection, we designed and implemented a scheme for identifying ES cells that are resistant to retroviral infection and subsequent cloning of the mutated gene.
A library of mutant ES cells was established by genome-wide insertional mutagenesis in Blm-deficient ES cells, and a screen was performed by superinfection of the library at high multiplicity with a recombinant retrovirus carrying a positive and negative selection cassette. Stringent negative selection was then used to exclude the infected ES cells. We successfully recovered five independent clones of ES cells that are resistant to retroviral infection. Analysis of the mutations in these clones revealed four different homozygous and one compound heterozygous mutation in the mCat-1 locus, which confirms that mCat-1 is the ecotropic murine leukemia virus receptor in ES cells.
We have demonstrated the feasibility and reliability of this recessive genetic approach to identifying critical genes required for retroviral infection in ES cells; the approach provides a unique opportunity to recover other cellular factors required for retroviral infection. The resulting insertionally mutated Blm-deficient ES cell library might also provide access to essential host cell components that are required for infection and replication for other types of virus.
PMCID: PMC1895998  PMID: 17407599
6.  The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery 
Retroviral vector-mediated gene transfer has been central to the development of gene therapy. Retroviruses have several distinct advantages over other vectors, especially when permanent gene transfer is the preferred outcome. The most important advantage that retroviral vectors offer is their ability to transform their single stranded RNA genome into a double stranded DNA molecule that stably integrates into the target cell genome. This means that retroviral vectors can be used to permanently modify the host cell nuclear genome. Recently, retroviral vector-mediated gene transfer, as well as the broader gene therapy field, has been re-invigorated with the development of a new class of retroviral vectors which are derived from lentiviruses. These have the unique ability amongst retroviruses of being able to infect non-cycling cells. Vectors derived from lentiviruses have provided a quantum leap in technology and seemingly offer the means to achieve significant levels of gene transfer in vivo.
The ability of retroviruses to integrate into the host cell chromosome also raises the possibility of insertional mutagenesis and oncogene activation. Both these phenomena are well known in the interactions of certain types of wild-type retroviruses with their hosts. However, until recently they had not been observed in replication defective retroviral vector-mediated gene transfer, either in animal models or in clinical trials. This has meant the potential disadvantages of retroviral mediated gene therapy have, until recently, been seen as largely, if not entirely, hypothetical. The recent clinical trial of γc mediated gene therapy for X-linked severe combined immunodeficiency (X-SCID) has proven the potential of retroviral mediated gene transfer for the treatment of inherited metabolic disease. However, it has also illustrated the potential dangers involved, with 2 out of 10 patients developing T cell leukemia as a consequence of the treatment. A considered review of retroviral induced pathogenesis suggests these events were qualitatively, if not quantitatively, predictable. In addition, it is clear that the probability of such events can be greatly reduced by relatively simple vector modifications, such as the use of self-inactivating vectors and vectors derived from non-oncogenic retroviruses. However, these approaches remain to be fully developed and validated. This review also suggests that, in all likelihood, there are no other major retroviral pathogenetic mechanisms that are of general relevance to replication defective retroviral vectors. These are important conclusions as they suggest that, by careful design and engineering of retroviral vectors, we can continue to use this gene transfer technology with confidence.
PMCID: PMC515179  PMID: 15310406
7.  TILLING - a shortcut in functional genomics 
Journal of Applied Genetics  2011;52(4):371-390.
Recent advances in large-scale genome sequencing projects have opened up new possibilities for the application of conventional mutation techniques in not only forward but also reverse genetics strategies. TILLING (Targeting Induced Local Lesions IN Genomes) was developed a decade ago as an alternative to insertional mutagenesis. It takes advantage of classical mutagenesis, sequence availability and high-throughput screening for nucleotide polymorphisms in a targeted sequence. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of its genome size and ploidy level. The TILLING protocol provides a high frequency of point mutations distributed randomly in the genome. The great mutagenic potential of chemical agents to generate a high rate of nucleotide substitutions has been proven by the high density of mutations reported for TILLING populations in various plant species. For most of them, the analysis of several genes revealed 1 mutation/200–500 kb screened and much higher densities were observed for polyploid species, such as wheat. High-throughput TILLING permits the rapid and low-cost discovery of new alleles that are induced in plants. Several research centres have established a TILLING public service for various plant species. The recent trends in TILLING procedures rely on the diversification of bioinformatic tools, new methods of mutation detection, including mismatch-specific and sensitive endonucleases, but also various alternatives for LI-COR screening and single nucleotide polymorphism (SNP) discovery using next-generation sequencing technologies. The TILLING strategy has found numerous applications in functional genomics. Additionally, wide applications of this throughput method in basic and applied research have already been implemented through modifications of the original TILLING strategy, such as Ecotilling or Deletion TILLING.
PMCID: PMC3189332  PMID: 21912935
TILLING platform; Plant mutagenesis; Bioinformatic tools; Reverse genetics; Next-generation sequencing technologies
8.  piggyBac Transposon Somatic Mutagenesis with an Activated Reporter and Tracker (PB-SMART) for Genetic Screens in Mice 
PLoS ONE  2011;6(10):e26650.
Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.
PMCID: PMC3198810  PMID: 22039523
9.  New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis 
BMC Genomics  2013;14:786.
In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions INGenomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported.
In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries.
These results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.
PMCID: PMC3830513  PMID: 24225309
TILLING; Fugu; ENU; HRM; Myostatin; Mutagenesis; Fish breeding
10.  RTCGD: retroviral tagged cancer gene database 
Nucleic Acids Research  2004;32(Database issue):D523-D527.
Retroviral insertional mutagenesis in mouse hematopoietic tumors provides a potent cancer gene discovery tool in the post-genome-sequence era. To manage multiple high-throughput insertional mutagenesis screening projects, we developed the Retroviral Tagged Cancer Gene Database (RTCGD; A sequence analysis pipeline determines the genomic position of each retroviral integration site cloned from a mouse tumor, the distance between it and the nearest candidate disease gene(s) and its orientation with respect to the candidate gene(s). The pipeline also identifies genomic regions that are targets of retroviral integration in more than one tumor (common integration sites, CISs) and are thus likely to encode a disease gene. Users can search the database using a specified gene symbol, chromosome number or tumor model to identify both CIS genes and unique viral integration sites or compare the integration sites cloned by different laboratories using different models. As a default setting, users first review the CIS Lists and then Clone Lists. CIS Lists describe CISs and their candidate disease genes along with links to other public databases and clone lists. Clone Lists describe the viral integration site clones along with the tumor model and tumor type from which they were cloned, candidate disease gene(s), genomic position and orientation of the integrated provirus with respect to the candidate gene(s). It also provides a pictorial view of the genomic location of each integration site relative to neighboring genes and markers. Researchers can identify integrations of interest and compare their results with those for multiple tumor models and tumor types using RTCGD.
PMCID: PMC308748  PMID: 14681473
11.  Hematopoietic stem cells, hematopoiesis and disease: lessons from the zebrafish model 
Genome Medicine  2011;3(12):83.
The zebrafish model is rapidly gaining prominence in the study of development, hematopoiesis, and disease. The zebrafish provides distinct advantages over other vertebrate models during early embryonic development by producing transparent, externally fertilized embryos. Embryonic zebrafish are easily visualized and manipulated through microinjection, chemical treatment, and mutagenesis. These procedures have contributed to large-scale chemical, suppressor, and genetic screens to identify hematopoietic gene mutations. Genomic conservation and local synteny between the human and zebrafish genomes make genome-scale and epigenetic analysis of these mutations (by microarray, chromatin immunoprecipitation sequencing, and RNA sequencing procedures) powerful methods for translational research and medical discovery. In addition, large-scale screening techniques have resulted in the identification of several small molecules capable of rescuing hematopoietic defects and inhibiting disease. Here, we discuss the contributions of the zebrafish model to the understanding of hematopoiesis, hematopoietic stem cell development, and disease-related discovery. We also highlight the recent discovery of small molecules with clinical promise, such as dimethyl prostaglandin E2, 3F8, and thiazole-carboxamide 10A.
PMCID: PMC3334548  PMID: 22206610
Chemical screen, disease; fate mapping; hematopoiesis; HSCs; morpholino; mutagenesis; suppressor screen; transplantation; zebrafish
12.  Chemical Mutagens, Transposons, and Transgenes to Interrogate Gene Function in Drosophila melanogaster 
Methods (San Diego, Calif.)  2014;68(1):15-28.
The study of genetics, genes, and chromosomal inheritance was initiated by Thomas Morgan in when the first visible mutations were identified in fruit flies. The field expanded upon the work initiated by Herman Muller in 1926 when he used X-rays to develop the first balancer chromosomes. Today, balancers are still invaluable to maintain mutations and transgenes but the arsenal of tools has expanded vastly and numerous new methods have been developed, many relying on the availability of the genome sequence and transposable elements. Forward genetic screens based on chemical mutagenesis or transposable elements have resulted in the unbiased identification of many novel players involved in processes probed by specific phenotypic assays. Reverse genetic approaches have relied on the availability of a carefully selected set of transposon insertions spread throughout the genome to allow the manipulation of the region in the vicinity of each insertion. Lastly, the ability to transform Drosophila with single copy transgenes using transposons or site-specific integration using the ΦC31 integrase has allowed numerous manipulations, including the ability to create and integrate genomic rescue constructs, generate duplications, RNAi knock-out technology, binary expression systems like the GAL4/UAS system as well as other methods. Here, we will discuss the most useful methodologies to interrogate the fruit fly genome in vivo focusing on chemical mutagenesis, transposons and transgenes. Genome engineering approaches based on nucleases and RNAi technology are discussed in following chapters.
PMCID: PMC4061744  PMID: 24583113
13.  Insertional mutagenesis in mice deficient for p15Ink4b, p16Ink4a, p21Cip1, p27Kip1 reveals cancer gene interactions and correlations with tumor phenotypes 
Cancer research  2010;70(2):520-531.
The cyclin dependent kinase (CDK) inhibitors p15, p16, p21 and p27 are frequently deleted, silenced or downregulated in many malignancies. Inactivation of CDK inhibitors predisposes mice to tumor development demonstrating that these genes can act as tumor suppressors. Here we describe high-throughput murine leukemia virus (MuLV) insertional mutagenesis screens in mice deficient for one or a combination of two CDK inhibitors. We retrieved 9117 retroviral insertions from 476 lymphomas and find hundreds of loci that are mutated significantly more frequently than expected by chance. Many of these are skewed toward a specific genetic context of predisposing germline and somatic mutations. We also find associations between these loci and gender, age of tumor onset and with lymphocyte lineage (B or T cell). Comparison of retroviral insertion sites with SNPs associated with chronic lymphocytic leukemia (CLL) reveals significant overlap between these datasets. Together these data highlight the importance of genetic context within large-scale mutation detection studies and demonstrate a novel use for insertional mutagenesis data in prioritization of disease associated genes resulting from genome-wide association studies.
PMCID: PMC2875110  PMID: 20068150
CDK inhibitors; insertional mutagenesis; lymphoma; CLL; Down syndrome
14.  Concise Review: Lessons Learned From Clinical Trials of Gene Therapy in Monogenic Immunodeficiency Diseases 
Thirty years ago, retroviral transfer of genetic material into hematopoietic stem and progenitor cells led to predictions that this technology would transform modern medicine. Lessons from adverse events have now led to a new generation of vectors, refinements in conditioning regimens, and manufacturing, which are paving the way for expanded applications of the current technology and recent emphasis on gene targeting/genome editing as the next advancements in the field.
Thirty years ago, retroviral transfer of genetic material into hematopoietic stem and progenitor cells (HSC/Ps) led to predictions that this technology would transform modern medicine [Nature 1983;305:556–558; Nature 1984;310:476–480]. Studies in several immunodeficiency diseases in the past 15 years have demonstrated clear proof of principle that gene therapy can have long-lasting, potentially curative effects without the need to search for allogeneic donors and without risk of graft-versus-host disease. Improvement in gene transfer efficiency for target HSC/Ps brought to light issues of insertional mutagenesis caused by transfer vectors, resulting in oncogene transactivation and leukemias. Lessons from these adverse events have now led to a new generation of vectors, refinements in conditioning regimens, and manufacturing, which are paving the way for expanded applications of the current technology and recent emphasis on gene targeting/genome editing as the next advancements in the field.
PMCID: PMC4006494  PMID: 24682287
Clinical trials; Gene therapy; Immunodeficiency; Hematopoietic cells; Progenitor cells
15.  Gene Mutations and Genomic Rearrangements in the Mouse as a Result of Transposon Mobilization from Chromosomal Concatemers 
PLoS Genetics  2006;2(9):e156.
Previous studies of the Sleeping Beauty (SB) transposon system, as an insertional mutagen in the germline of mice, have used reverse genetic approaches. These studies have led to its proposed use for regional saturation mutagenesis by taking a forward-genetic approach. Thus, we used the SB system to mutate a region of mouse Chromosome 11 in a forward-genetic screen for recessive lethal and viable phenotypes. This work represents the first reported use of an insertional mutagen in a phenotype-driven approach. The phenotype-driven approach was successful in both recovering visible and behavioral mutants, including dominant limb and recessive behavioral phenotypes, and allowing for the rapid identification of candidate gene disruptions. In addition, a high frequency of recessive lethal mutations arose as a result of genomic rearrangements near the site of transposition, resulting from transposon mobilization. The results suggest that the SB system could be used in a forward-genetic approach to recover interesting phenotypes, but that local chromosomal rearrangements should be anticipated in conjunction with single-copy, local transposon insertions in chromosomes. Additionally, these mice may serve as a model for chromosome rearrangements caused by transposable elements during the evolution of vertebrate genomes.
Perhaps the greatest challenge for biomedical research in the post-genomics era will be to assign functions to the human set of ~25,000 genes. The classical method for discovering the gene function is mutation. Thus, technologies that can mutate genes in mammalian genetic models like the mouse are under development in hopes of creating an efficient method to complete this task. One such technology, the Sleeping Beauty (SB) transposon system, was developed for this purpose in 2001. This mobile DNA element is highly active in transgenic mice and has been shown to disrupt mouse genes efficiently. Geurts et al. describe a novel attempt to use the SB transposon in a forward-genetic screen using an insertional mutagen, the first attempt of its kind. They discovered that the process of transposon mobilization in mouse chromosomes can lead to dramatic effects on local genomic sequences. Indeed, transposons like SB can cause genomic rearrangements including deletions, inversions and translocations, involving tens of thousands to tens of millions of base pairs. This discovery has important implications for using transposable elements for mouse germline mutagenesis and, at the same time, may provide a model for studying genomic rearrangements that have helped shape vertebrate genomes during evolution.
PMCID: PMC1584263  PMID: 17009875
16.  The essential genome of a bacterium 
This study reports the essential Caulobacter genome at 8 bp resolution determined by saturated transposon mutagenesis and high-throughput sequencing. This strategy is applicable to full genome essentiality studies in a broad class of bacterial species.
The essential Caulobacter genome was determined at 8 bp resolution using hyper-saturated transposon mutagenesis coupled with high-throughput sequencing.Essential protein-coding sequences comprise 90% of the essential genome; the remaining 10% comprising essential non-coding RNA sequences, gene regulatory elements and essential genome replication features.Of the 3876 annotated open reading frames (ORFs), 480 (12.4%) were essential ORFs, 3240 (83.6%) were non-essential ORFs and 156 (4.0%) were ORFs that severely impacted fitness when mutated.The essential elements are preferentially positioned near the origin and terminus of the Caulobacter chromosome.This high-resolution strategy is applicable to high-throughput, full genome essentiality studies and large-scale genetic perturbation experiments in a broad class of bacterial species.
The regulatory events that control polar differentiation and cell-cycle progression in the bacterium Caulobacter crescentus are highly integrated, and they have to occur in the proper order (McAdams and Shapiro, 2011). Components of the core regulatory circuit are largely known. Full discovery of its essential genome, including non-coding, regulatory and coding elements, is a prerequisite for understanding the complete regulatory network of this bacterial cell. We have identified all the essential coding and non-coding elements of the Caulobacter chromosome using a hyper-saturated transposon mutagenesis strategy that is scalable and can be readily extended to obtain rapid and accurate identification of the essential genome elements of any sequenced bacterial species at a resolution of a few base pairs.
We engineered a Tn5 derivative transposon (Tn5Pxyl) that carries at one end an inducible outward pointing Pxyl promoter (Christen et al, 2010). We showed that this transposon construct inserts into the genome randomly where it can activate or disrupt transcription at the site of integration, depending on the insertion orientation. DNA from hundred of thousands of transposon insertion sites reading outward into flanking genomic regions was parallel PCR amplified and sequenced by Illumina paired-end sequencing to locate the insertion site in each mutant strain (Figure 1). A single sequencing run on DNA from a mutagenized cell population yielded 118 million raw sequencing reads. Of these, >90 million (>80%) read outward from the transposon element into adjacent genomic DNA regions and the insertion site could be mapped with single nucleotide resolution. This yielded the location and orientation of 428 735 independent transposon insertions in the 4-Mbp Caulobacter genome.
Within non-coding sequences of the Caulobacter genome, we detected 130 non-disruptable DNA segments between 90 and 393 bp long in addition to all essential promoter elements. Among 27 previously identified and validated sRNAs (Landt et al, 2008), three were contained within non-disruptable DNA segments and another three were partially disruptable, that is, insertions caused a notable growth defect. Two additional small RNAs found to be essential are the transfer-messenger RNA (tmRNA) and the ribozyme RNAseP (Landt et al, 2008). In addition to the 8 non-disruptable sRNAs, 29 out of the 130 intergenic essential non-coding sequences contained non-redundant tRNA genes; duplicated tRNA genes were non-essential. We also identified two non-disruptable DNA segments within the chromosomal origin of replication. Thus, we resolved essential non-coding RNAs, tRNAs and essential replication elements within the origin region of the chromosome. An additional 90 non-disruptable small genome elements of currently unknown function were identified. Eighteen of these are conserved in at least one closely related species. Only 2 could encode a protein of over 50 amino acids.
For each of the 3876 annotated open reading frames (ORFs), we analyzed the distribution, orientation, and genetic context of transposon insertions. There are 480 essential ORFs and 3240 non-essential ORFs. In addition, there were 156 ORFs that severely impacted fitness when mutated. The 8-bp resolution allowed a dissection of the essential and non-essential regions of the coding sequences. Sixty ORFs had transposon insertions within a significant portion of their 3′ region but lacked insertions in the essential 5′ coding region, allowing the identification of non-essential protein segments. For example, transposon insertions in the essential cell-cycle regulatory gene divL, a tyrosine kinase, showed that the last 204 C-terminal amino acids did not impact viability, confirming previous reports that the C-terminal ATPase domain of DivL is dispensable for viability (Reisinger et al, 2007; Iniesta et al, 2010). In addition, we found that 30 out of 480 (6.3%) of the essential ORFs appear to be shorter than the annotated ORF, suggesting that these are probably mis-annotated.
Among the 480 ORFs essential for growth on rich media, there were 10 essential transcriptional regulatory proteins, including 5 previously identified cell-cycle regulators (McAdams and Shapiro, 2003; Holtzendorff et al, 2004; Collier and Shapiro, 2007; Gora et al, 2010; Tan et al, 2010) and 5 uncharacterized predicted transcription factors. In addition, two RNA polymerase sigma factors RpoH and RpoD, as well as the anti-sigma factor ChrR, which mitigates rpoE-dependent stress response under physiological growth conditions (Lourenco and Gomes, 2009), were also found to be essential. Thus, a set of 10 transcription factors, 2 RNA polymerase sigma factors and 1 anti-sigma factor are the core essential transcriptional regulators for growth on rich media. To further characterize the core components of the Caulobacter cell-cycle control network, we identified all essential regulatory sequences and operon transcripts. Altogether, the 480 essential protein-coding and 37 essential RNA-coding Caulobacter genes are organized into operons such that 402 individual promoter regions are sufficient to regulate their expression. Of these 402 essential promoters, the transcription start sites (TSSs) of 105 were previously identified (McGrath et al, 2007).
The essential genome features are non-uniformly distributed on the Caulobacter genome and enriched near the origin and the terminus regions. In contrast, the chromosomal positions of the published E. coli essential coding sequences (Rocha, 2004) are preferentially located at either side of the origin (Figure 4A). This indicates that there are selective pressures on chromosomal positioning of some essential elements (Figure 4A).
The strategy described in this report could be readily extended to quickly determine the essential genome for a large class of bacterial species.
Caulobacter crescentus is a model organism for the integrated circuitry that runs a bacterial cell cycle. Full discovery of its essential genome, including non-coding, regulatory and coding elements, is a prerequisite for understanding the complete regulatory network of a bacterial cell. Using hyper-saturated transposon mutagenesis coupled with high-throughput sequencing, we determined the essential Caulobacter genome at 8 bp resolution, including 1012 essential genome features: 480 ORFs, 402 regulatory sequences and 130 non-coding elements, including 90 intergenic segments of unknown function. The essential transcriptional circuitry for growth on rich media includes 10 transcription factors, 2 RNA polymerase sigma factors and 1 anti-sigma factor. We identified all essential promoter elements for the cell cycle-regulated genes. The essential elements are preferentially positioned near the origin and terminus of the chromosome. The high-resolution strategy used here is applicable to high-throughput, full genome essentiality studies and large-scale genetic perturbation experiments in a broad class of bacterial species.
PMCID: PMC3202797  PMID: 21878915
functional genomics; next-generation sequencing; systems biology; transposon mutagenesis
17.  Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis 
mBio  2016;7(1):e01817-15.
Although progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP) and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR) from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation.
The obligate intracellular nature of a variety of infectious bacteria presents a significant obstacle to the development of molecular genetic tools for dissecting pathogenicity. Although progress in chlamydial genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Here, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis L2. Furthermore, this approach permits monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms.
PMCID: PMC4725004  PMID: 26787828
18.  Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis 
PLoS Computational Biology  2016;12(9):e1005074.
Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap) host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase). Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B) identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline) that may be potential for antiviral indication (e.g. anti-Ebola). In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics.
Author Summary
Infectious diseases result in millions of deaths and cost billions of dollars annually. Hence, there is urgency for developing more innovative and effective antiviral therapeutics. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses. We herein reported over 700 candidate cellular genes, over 20% of which were independently selected by multiple viruses in one or more cell types. Using systems biology-based analysis, we found that host genes associated with viral replication tended to occupy central hubs in the human protein interactome and to be ancient genes with low evolutionary rates, compared to non-virus-associated genes. Cell cycle phase-specific sub-network analysis showed that host cell cycle program played important roles during viral replication by regulating specific cell cycle phases. Moreover, we presented novel evidences to suggest that host genes supporting viral replication were frequently implicated in Mendelian and orphan diseases, or played critical roles in cancer. Importantly, we found approximately 110 new putative druggable antiviral targets by merging genome-wide gene-trap insertional mutagenesis, drug-gene network, and bioinformatics data. Furthermore, we have demonstrated the use of a computable representation of genetic testing to effectively identify new potential antiviral indications for existing drugs. In summary, this study presents new and important methodologies for developing broadly active antiviral therapeutics.
PMCID: PMC5025164  PMID: 27632082
19.  The Optimal Mutagen Dosage to Induce Point-Mutations in Synechocystis sp. PCC6803 and Its Application to Promote Temperature Tolerance 
PLoS ONE  2012;7(11):e49467.
Random mutagenesis is a useful tool to genetically modify organisms for various purposes, such as adaptation to cultivation conditions, the induction of tolerances, or increased yield of valuable substances. This is especially attractive for systems where it is not obvious which genes require modifications. Random mutagenesis has been extensively used to modify crop plants, but even with the renewed interest in microalgae and cyanobacteria for biofuel applications, there is relatively limited current research available on the application of random mutagenesis for these organisms, especially for cyanobacteria. In the presented work we characterized the lethality and rate of non-lethal point mutations for ultraviolet radiation and methyl methanesulphonate on the model cyanobacteria Synechocystis sp. PCC6803. Based on these results an optimal dosage of 10–50 J/m2 for UV and either 0.1 or 1 v% for MMS was determined. A Synechocystis wildtype culture was then mutagenized and selected for increased temperature tolerance in vivo. During the second round of mutagenesis the viability of the culture was monitored on a cell by cell level from the treatment of the cells up to the growth at an increased temperature. After four distinct rounds of treatment (two with each mutagen) the temperature tolerance of the strain was effectively raised by about 2°C. Coupled with an appropriate in vivo screening, the described methods should be applicable to induce a variety of desirable characteristics in various strains. Coupling random mutagenesis with high-throughput screening methods would additionally allow to select for important characteristics for biofuel production, which do not yield a higher fitness and can not be selected for in vivo, such as fatty acid concentration. In a combined approach with full genome sequencing random mutagenesis could be used to determine suitable target-genes for more focused methods.
PMCID: PMC3504032  PMID: 23185339
20.  A PATO-compliant zebrafish screening database (MODB): management of morpholino knockdown screen information 
BMC Bioinformatics  2008;9:7.
The zebrafish is a powerful model vertebrate amenable to high throughput in vivo genetic analyses. Examples include reverse genetic screens using morpholino knockdown, expression-based screening using enhancer trapping and forward genetic screening using transposon insertional mutagenesis. We have created a database to facilitate web-based distribution of data from such genetic studies.
The MOrpholino DataBase is a MySQL relational database with an online, PHP interface. Multiple quality control levels allow differential access to data in raw and finished formats. MODBv1 includes sequence information relating to almost 800 morpholinos and their targets and phenotypic data regarding the dose effect of each morpholino (mortality, toxicity and defects). To improve the searchability of this database, we have incorporated a fixed-vocabulary defect ontology that allows for the organization of morpholino affects based on anatomical structure affected and defect produced. This also allows comparison between species utilizing Phenotypic Attribute Trait Ontology (PATO) designated terminology. MODB is also cross-linked with ZFIN, allowing full searches between the two databases. MODB offers users the ability to retrieve morpholino data by sequence of morpholino or target, name of target, anatomical structure affected and defect produced.
MODB data can be used for functional genomic analysis of morpholino design to maximize efficacy and minimize toxicity. MODB also serves as a template for future sequence-based functional genetic screen databases, and it is currently being used as a model for the creation of a mutagenic insertional transposon database.
PMCID: PMC2221974  PMID: 18179718
21.  3D hotspots of recurrent retroviral insertions reveal long-range interactions with cancer genes 
Nature Communications  2015;6:6381.
Genomically distal mutations can contribute to the deregulation of cancer genes by engaging in chromatin interactions. To study this, we overlay viral cancer-causing insertions obtained in a murine retroviral insertional mutagenesis screen with genome-wide chromatin conformation capture data. Here we find that insertions tend to cluster in 3D hotspots within the nucleus. The identified hotspots are significantly enriched for known cancer genes, and bear the expected characteristics of bona fide regulatory interactions, such as enrichment for transcription factor-binding sites. In addition, we observe a striking pattern of mutual exclusive integration. This is an indication that insertions in these loci target the same gene, either in their linear genomic vicinity or in their 3D spatial vicinity. Our findings shed new light on the repertoire of targets obtained from insertional mutagenesis screening and underline the importance of considering the genome as a 3D structure when studying effects of genomic perturbations.
Retroviral insertional mutagenesis is used for identifying genes involved in the development of cancer. Here, the authors overlay cancer-causing insertions with genome-wide Hi-C data and find that retroviral elements tend to cluster in 3D hotspots.
PMCID: PMC4351571  PMID: 25721899
22.  Enhanced gene expression from retroviral vectors 
BMC Biotechnology  2008;8:19.
Retroviruses are widely used to transfer genes to mammalian cells efficiently and stably. However, genetic elements required for high-level gene expression are incompatible with standard systems. The retroviral RNA genome is produced by cellular transcription and post-transcriptional processing within packaging cells: Introns present in the retroviral genomic transcript are removed by splicing, while polyadenylation signals lead to the production of ineffective truncated genomes. Furthermore strong enhancer/promoters within the retroviral payload lead to detrimental competition with the retroviral enhancer/promoter.
By exploiting a new method of producing the retroviral genome in vitro it is possible to produce infectious retroviral particles carrying a high-level expression cassette that completely prohibits production of infectious retroviral particles by conventional methods.
We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron, the GFP open reading frame and a strong polyadenylation signal. This cassette was cloned into both a conventional MMLV retroviral vector and a vector designed to allow in vitro transcription of the retroviral genome by T7 RNA polymerase.
When the conventional retroviral vector was transfected into packaging cells, the expression cassette drove strong GFP expression, but no infectious retrovirus was produced. Introduction of the in vitro produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression. However, infectious retrovirus was easily recovered, and when used to infect target primary human cells led to very high GFP expression – up to 3.5 times greater than conventional retroviral LTR-driven expression.
Retroviral vectors carrying an optimized high-level expression cassette do not produce infectious virions when introduced into packaging cells by transfection of DNA. Infectious retrovirus carrying the same cassette is readily produced when packaging cells are transfected with in vitro transcribed retroviral genomic RNA. The applications of this technique are not limited to producing the higher levels of transgene expression demonstrated here. For example, novel reporters with alternatively spliced exon-intron configurations could readily be transduced into virtually any cell. Furthermore, because the in vitro transcripts are not translated within the packaging cells, retroviruses carrying genes lethal to the packaging cells can also be produced.
PMCID: PMC2266726  PMID: 18298815
23.  Sleeping Beauty Transposon Mutagenesis of the Rat Genome in Spermatogonial Stem Cells 
Methods (San Diego, Calif.)  2010;53(4):356-365.
Since several aspects of physiology in rats has evolved to be more similar to humans than that of mice, it is highly desirable to link the rat into the process of annotating the human genome with function. However, the lack of technology for generating defined mutants in the rat genome has hindered the identification of causative relationships between genes and disease phenotypes. As an important step towards this goal, an approach of establishing transposon-mediated insertional mutagenesis in rat spermatogonial stem cells was recently developed. Transposons can be viewed as natural DNA transfer vehicles that, similar to integrating viruses, are capable of efficient genomic insertion. The mobility of transposons can be controlled by conditionally providing the transposase component of the transposition reaction. Thus, a DNA of interest such as a mutagenic gene trap cassette cloned between the inverted repeat sequences of a transposon-based vector can be utilized for stable genomic insertion in a regulated and highly efficient manner. Gene trap transposons integrate into the genome in a random fashion, and those mutagenic insertions that occurred in expressed genes can be selected in vitro based on activation of a reporter. Selected monoclonal as well as polyclonal libraries of gene trap clones are transplanted into the testes of recipient/founder male rats allowing passage of the mutation through the germline to F1 progeny after only a single cross with wild-type females. This paradigm enables a powerful methodological pipeline for forward genetic screens for functional gene annotation in the rat, as well as other vertebrate models. This article provides a detailed description on how to culturerat spermatogonial stem cell lines, their transfection with transposon plasmids, selection of gene trap insertions with antibiotics, transplantation of genetically modified stem cells and genotyping of knockout animals.
PMCID: PMC3164264  PMID: 21193047
24.  EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations 
Plant Methods  2014;10:5.
TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that combines chemical mutagenesis with high-throughput genome-wide screening for point mutation detection in genes of interest. However, this mutation discovery approach faces a particular problem which is how to obtain a mutant population with a sufficiently high mutation density. Furthermore, plant mutagenesis protocols require two successive generations (M1, M2) for mutation fixation to occur before the analysis of the genotype can begin.
Here, we describe a new TILLING approach for rice based on ethyl methanesulfonate (EMS) mutagenesis of mature seed-derived calli and direct screening of in vitro regenerated plants. A high mutagenesis rate was obtained (i.e. one mutation in every 451 Kb) when plants were screened for two senescence-related genes. Screening was carried out in 2400 individuals from a mutant population of 6912. Seven sense change mutations out of 15 point mutations were identified.
This new strategy represents a significant advantage in terms of time-savings (i.e. more than eight months), greenhouse space and work during the generation of mutant plant populations. Furthermore, this effective chemical mutagenesis protocol ensures high mutagenesis rates thereby saving in waste removal costs and the total amount of mutagen needed thanks to the mutagenesis volume reduction.
PMCID: PMC3923009  PMID: 24475756
Oryza sativa; Scutellum; Mature seed; Calli; Mutagenesis; EMS; TILLING; Reverse genetics
25.  Common Sites of Retroviral Integration in Mouse Hematopoietic Tumors Identified by High-Throughput, Single Nucleotide Polymorphism-Based Mapping and Bacterial Artificial Chromosome Hybridization 
Journal of Virology  2003;77(2):1584-1588.
Retroviral insertional mutagenesis in mouse hematopoietic tumors provides a powerful cancer gene discovery tool. Here, we describe a high-throughput, single nucleotide polymorphism (SNP)-based method, for mapping retroviral integration sites cloned from mouse tumors, and a bacterial artificial chromosome (BAC) hybridization method, for localizing these retroviral integration sites to common sites of retroviral integration (CISs). Several new CISs were identified, including one CIS that mapped near Notch1, a gene that has been causally associated with human T-cell tumors. This mapping method is applicable to many different species, including ones where few genetic markers and little genomic sequence information are available. It can also be used to map endogenous proviruses.
PMCID: PMC140841  PMID: 12502872

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