This document discusses insertional mutagenesis, specifically focusing on insertional mutagenesis. It defines insertional mutagenesis as the integration of exogenous DNA into a host genome, which can deregulate nearby genes and alter cellular phenotype. Retroviruses and transposons are commonly used as integrating agents in insertional mutagenesis experiments to identify novel cancer genes. The document describes how retroviruses like MoMLV and MMTV integrate randomly into the host genome and how analyzing common insertion sites from tumors can reveal cancer-causing genes. It also explains the different mechanisms of insertional mutagenesis, such as enhancer insertion, promoter insertion, and intragenic insertion, and how each can alter gene expression
This presentation discusses marker assisted selection (MAS), a method for indirect plant breeding selection. MAS uses molecular markers linked to traits of interest, like disease resistance or yield, to select plants without observing the trait itself. The presentation defines MAS and different types of molecular markers like RFLPs, SSLPs, AFLPs. It outlines the steps of MAS, including selecting parents, developing breeding populations, isolating DNA, scoring markers, and correlating markers with traits. Benefits of MAS include high accuracy, allowing selection of traits affected by environment. Examples of using MAS in crops like barley, maize, rice and wheat are also provided.
This document discusses several methods for studying gene function, including classical approaches like loss of function and gain of function mutations. It then summarizes various applications of activation tagging, such as discovering disease resistance genes in Arabidopsis and identifying metabolites in Catharanthus roseus. The document also describes fruit color and shape mutants identified from activation tagging in tomato plants, and recent advances studying drought tolerance genes using activation tagging mutants in tomato. Specific mutants and tagged genes are discussed, including their differential expression patterns and involvement in processes like transcription, ribosome biogenesis, and protein ubiquitination. Both advantages and disadvantages of activation tagging are listed.
Genetic mapping is based on recombination frequencies between genetic loci during meiosis. Physical mapping determines the actual distances in base pairs between sequences on a chromosome using overlapping DNA fragments. Before whole genome sequencing, physical maps were created using techniques like restriction mapping of large-insert clones, probing genomic libraries with end fragments, and chromosome walking to build contigs of overlapping sequences. This allowed sequencing of individual fragments which could then be assembled into a complete genome sequence.
Map-based cloning is a technique used to identify the genetic cause of a mutant phenotype by isolating overlapping DNA segments that progress along the chromosome toward a candidate gene. The process involves initially identifying a marker close to the gene of interest and then saturating the region with additional markers. Large populations are screened to find markers that rarely recombine with the gene. Genomic libraries are screened to find clones containing the markers, and chromosomal walking is used to obtain flanking markers on a single clone. DNA fragments between the markers are tested to rescue the wild-type phenotype and identify the candidate gene.
This document discusses strategies for genome-wide mutagenesis. It describes three main strategies: transposon insertion, gene disruption through allelic exchange, and expression inhibition using antisense RNA. Transposon insertion involves using transposable elements to randomly insert into genomes. Gene disruption uses targeted homologous recombination to replace genes. Antisense RNA inhibits gene expression by binding to target mRNA. The document also discusses various methods for detecting mutations, such as single-strand conformation polymorphism and allele-specific oligonucleotide hybridization.
Virus-induced gene silencing (VIGS) is a technique that uses a viral vector carrying a fragment of a plant gene of interest to induce gene silencing. When the virus infects the plant, double-stranded RNA is produced from the viral genome and plant gene fragment, which is cleaved by the plant's dicer enzyme into siRNAs. These siRNAs are incorporated into the RISC complex and guide mRNA degradation of the targeted endogenous gene. Examples discussed were using tobacco rattle virus for VIGS in Nicotiana benthamiana and silencing tomato genes involved in the GABA pathway to study their roles in salt tolerance.
The document provides a history of genomics, beginning with Mendel's work in 1866 establishing the gene concept and laws of genetics. Key developments include the discovery of DNA in 1871, the chromosomal theory of inheritance in 1902, and the discovery of linkage in 1910. The structure of DNA was elucidated in the 1950s, leading to the development of recombinant DNA technology in the 1970s and DNA sequencing techniques in the 1970s-80s. The first whole genome of an organism was sequenced in 1995. More recent developments include next generation sequencing starting in 2005 and the advent of CRISPR/Cas9 genome editing in 2012. The document concludes with discussing the potential use of CRISPR to target genes in the diamondb
RNA splicing is the process by which introns, or non-coding sequences, are removed from pre-messenger RNA (pre-mRNA) to produce mature mRNA that can be translated into protein. Most genes contain introns that are removed by a spliceosome, a complex of RNA and proteins, leaving just the coding exons to form mRNA. Alternative splicing allows one gene to encode multiple proteins by selecting different combinations of exons. Errors in splicing can cause diseases if they result in truncated or abnormal proteins.
This presentation discusses marker assisted selection (MAS), a method for indirect plant breeding selection. MAS uses molecular markers linked to traits of interest, like disease resistance or yield, to select plants without observing the trait itself. The presentation defines MAS and different types of molecular markers like RFLPs, SSLPs, AFLPs. It outlines the steps of MAS, including selecting parents, developing breeding populations, isolating DNA, scoring markers, and correlating markers with traits. Benefits of MAS include high accuracy, allowing selection of traits affected by environment. Examples of using MAS in crops like barley, maize, rice and wheat are also provided.
This document discusses several methods for studying gene function, including classical approaches like loss of function and gain of function mutations. It then summarizes various applications of activation tagging, such as discovering disease resistance genes in Arabidopsis and identifying metabolites in Catharanthus roseus. The document also describes fruit color and shape mutants identified from activation tagging in tomato plants, and recent advances studying drought tolerance genes using activation tagging mutants in tomato. Specific mutants and tagged genes are discussed, including their differential expression patterns and involvement in processes like transcription, ribosome biogenesis, and protein ubiquitination. Both advantages and disadvantages of activation tagging are listed.
Genetic mapping is based on recombination frequencies between genetic loci during meiosis. Physical mapping determines the actual distances in base pairs between sequences on a chromosome using overlapping DNA fragments. Before whole genome sequencing, physical maps were created using techniques like restriction mapping of large-insert clones, probing genomic libraries with end fragments, and chromosome walking to build contigs of overlapping sequences. This allowed sequencing of individual fragments which could then be assembled into a complete genome sequence.
Map-based cloning is a technique used to identify the genetic cause of a mutant phenotype by isolating overlapping DNA segments that progress along the chromosome toward a candidate gene. The process involves initially identifying a marker close to the gene of interest and then saturating the region with additional markers. Large populations are screened to find markers that rarely recombine with the gene. Genomic libraries are screened to find clones containing the markers, and chromosomal walking is used to obtain flanking markers on a single clone. DNA fragments between the markers are tested to rescue the wild-type phenotype and identify the candidate gene.
This document discusses strategies for genome-wide mutagenesis. It describes three main strategies: transposon insertion, gene disruption through allelic exchange, and expression inhibition using antisense RNA. Transposon insertion involves using transposable elements to randomly insert into genomes. Gene disruption uses targeted homologous recombination to replace genes. Antisense RNA inhibits gene expression by binding to target mRNA. The document also discusses various methods for detecting mutations, such as single-strand conformation polymorphism and allele-specific oligonucleotide hybridization.
Virus-induced gene silencing (VIGS) is a technique that uses a viral vector carrying a fragment of a plant gene of interest to induce gene silencing. When the virus infects the plant, double-stranded RNA is produced from the viral genome and plant gene fragment, which is cleaved by the plant's dicer enzyme into siRNAs. These siRNAs are incorporated into the RISC complex and guide mRNA degradation of the targeted endogenous gene. Examples discussed were using tobacco rattle virus for VIGS in Nicotiana benthamiana and silencing tomato genes involved in the GABA pathway to study their roles in salt tolerance.
The document provides a history of genomics, beginning with Mendel's work in 1866 establishing the gene concept and laws of genetics. Key developments include the discovery of DNA in 1871, the chromosomal theory of inheritance in 1902, and the discovery of linkage in 1910. The structure of DNA was elucidated in the 1950s, leading to the development of recombinant DNA technology in the 1970s and DNA sequencing techniques in the 1970s-80s. The first whole genome of an organism was sequenced in 1995. More recent developments include next generation sequencing starting in 2005 and the advent of CRISPR/Cas9 genome editing in 2012. The document concludes with discussing the potential use of CRISPR to target genes in the diamondb
RNA splicing is the process by which introns, or non-coding sequences, are removed from pre-messenger RNA (pre-mRNA) to produce mature mRNA that can be translated into protein. Most genes contain introns that are removed by a spliceosome, a complex of RNA and proteins, leaving just the coding exons to form mRNA. Alternative splicing allows one gene to encode multiple proteins by selecting different combinations of exons. Errors in splicing can cause diseases if they result in truncated or abnormal proteins.
The plant nuclear genome consists of DNA organized into chromosomes within the cell nucleus. It contains both coding and regulatory sequences. The plant nuclear genome is made up of DNA, histone and non-histone proteins. DNA is packaged into nucleosomes containing histones and wrapped into chromatin. Chromatin exists in two forms - euchromatin which is loosely packaged and genetically active, and heterochromatin which is tightly packaged. Specific sequences like centromeres and telomeres aid in chromosome structure and integrity. The nuclear genome also contains both single-copy and repetitive non-coding DNA sequences.
This document provides information about physical mapping techniques used in molecular biology. It discusses that physical mapping can determine the sequence and physical distance between DNA base pairs with high accuracy. There are two main types of physical mapping: low resolution mapping, which can resolve DNA ranging from 1 base pair to several megabases, and high resolution mapping, which can resolve hundreds of kilobases to a single nucleotide. Some key techniques used for physical mapping include restriction mapping, fluorescent in situ hybridization (FISH) mapping, and sequence tagged site (STS) mapping. Restriction mapping involves cutting DNA at specific restriction sites to map fragment locations. FISH allows localization of specific DNA sequences on chromosomes using fluorescent probes. STS mapping uses short, unique
Molecular markers are DNA sequences that can be easily detected and whose inheritance can be monitored. They are based on natural polymorphisms and allow studying the inheritance of genes. Common types of molecular markers include RFLPs, RAPDs, AFLPs, SSRs, and SNPs. RFLPs use restriction enzymes to detect differences in fragment lengths. RAPDs use random primers to detect sequence polymorphisms. AFLPs selectively amplify restriction fragments to detect length differences. SSRs detect variability in simple sequence repeats. Molecular markers are useful for applications like gene mapping, phylogenetic studies, and analyzing genetic diversity.
This document discusses molecular breeding techniques using the Barnase-Barstar system for inducing male sterility in plants. It explains that the Barnase gene is cytotoxic and kills tapetum cells, preventing pollen development and resulting in transgenic male sterility. The Barstar gene provides fertility restoration. The system has been used successfully in tobacco and oilseed rape to develop hybrid seeds. Some benefits of this system include efficient fertility restoration, easy maintenance of male sterile lines, and elimination of male fertile plants from lines. However, alternative systems that are more attractive than Barnase-Barstar have also been explored.
This document discusses DNA sequencing methods. It describes the Maxam-Gilbert sequencing method developed in 1976-1977 which uses chemical modification and cleavage of DNA at specific bases, followed by electrophoresis to separate fragments by size. It also mentions the popular Sanger sequencing method. The procedure for Maxam-Gilbert sequencing involves labeling DNA, cleaving it with chemicals, running the fragments on a gel, and analyzing the results to deduce the DNA sequence. Advantages include no premature termination and ability to sequence stretches not possible with enzymatic methods, while disadvantages include use of radioactivity and toxic chemicals.
presented by HAFIZ M WASEEM
university of education LAHORE Pakistan
i am from mailsi vehari and studied in lahore
bsc in science college multan
msc from lahore
Introduction
Transcriptome analysis
Goal of functional genomics
Why we need functional genomics
Technique
1. At DNA level
2.At RNA level
3. At protein level
4. loss of function
5. functional genomic and bioinformatics
Application
Latest research and reviews
Websites of functional genomics
Conclusions
Reference
Sequence tagged sites (STSs) are short DNA sequences that can be used as genetic markers. STSs were introduced in 1989 as a way to map genes along chromosomes using PCR. They serve as landmarks on physical maps of genomes. STSs are mapped by breaking genomes into fragments, replicating the fragments in bacterial cells to create libraries, and using PCR to determine which fragments contain STSs. Different types of STS markers include microsatellites, SCARs, CAPs, and ISSRs, each of which has distinct characteristics and applications in genetic mapping, population studies, and other areas.
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
this presentation is about the molecular markers as we all know the molecular markers are the DNA sequences it can be easily detected and its inheritance is easily monitored.so the main basics of the molecular markers is the polymorphic nature so it can used as molecular markers.and this will gives you the idea about AFLP, RFLP, RAPD, SNPS,ETC.
Introduction
Backcross breeding & its types
Marker assisted breeding
Marker assisted backcross breeding (MABC)
Main strategies
Advantages over conventional breeding
Case studies
Future outlook
Conclusion
This document discusses different methods for genome sequencing and assembly, including restriction enzyme fingerprinting, marker sequences, and hybridization assays. It focuses on using marker sequences like sequence-tagged sites (STS), expressed sequence tags (ESTs), untranslated regions (UTRs), and single nucleotide polymorphisms (SNPs) to map genomes. Large-insert cloning vectors like BACs and PACs can be used with restriction enzyme fingerprinting and FPC software to assemble contigs and map genomes at a large scale. Marker sequences provide a dense set of physical markers to build accurate physical maps of genomes.
This document discusses cytoplasmic male sterility (CMS), a maternally inherited trait in plants where the plant is unable to produce functional pollen. CMS is caused by mitochondrial mutations or rearrangements that interfere with pollen development. Nuclear restorer genes can suppress CMS by interacting with the mitochondrial genes. CMS is used in hybrid seed production systems in many crops.
Gene silencing, also known as RNA interference, is a natural process in plants that evolved as a defense mechanism against viruses. Transgene silencing occurs when introduced transgenes are not expressed due to this silencing process. The first evidence of this was discovered in 1990 by R. Jorgensen in petunia plants, where both an introduced gene and endogenous gene were silenced. Gene silencing can occur at the transcriptional or post-transcriptional level through mechanisms like siRNA and microRNA production. Virus-induced gene silencing is a technique used to study gene function and develop virus-resistant plants by suppressing viral gene expression. Applications of gene silencing include developing disease-resistant crops and modifying plant traits.
Marker-assisted selection (MAS) is a plant breeding method that uses DNA markers to select for desirable traits. It allows breeders to select plants earlier in development compared to phenotypic selection. MAS has advantages like being unaffected by environment and ability to select for recessive traits, but may be more expensive initially than conventional methods. Careful analysis of costs and benefits is needed to determine if MAS is advantageous for a particular program over traditional breeding. MAS requires tightly linked markers, knowledge of marker-trait associations, and data management to be effective. A variety of MAS approaches exist like backcrossing, pyramiding, and combined MAS and phenotypic selection.
- β-glucuronidase (GUS) is a commonly used reporter gene in plant molecular biology and genetic engineering to indicate successful introduction of foreign DNA into cells.
- GUS expression can be detected through fluorometric or histochemical assays, allowing visualization of promoter activity, protein localization, and transgenic events.
- The GUS gene is fused to genes of interest, and GUS activity is used to study processes like tissue-specific expression, response to stresses, and transformation efficiency.
- While destructive, GUS is a stable and non-toxic reporter enabling versatile applications in fundamental and applied plant research.
This document describes a study that used T-DNA tagging to activate gene expression in rice. Researchers constructed vectors containing the CaMV 35S enhancer and introduced them into rice plants via Agrobacterium-mediated transformation. They generated over 13,000 transgenic lines and isolated sequences flanking the T-DNA insertions in 71 lines. They found that in 21 lines, the T-DNA inserted within 4.5kb of a gene, positioning the enhancer to potentially activate expression. Reverse transcription PCR revealed four lines where the nearby gene showed increased expression levels compared to wild-type, demonstrating the ability of T-DNA tagging to enhance gene expression.
The document discusses the C-value paradox, which is the lack of relationship between genome size and organism complexity. It provides data on the wide range of genome sizes across different taxonomic groups. Introns and exons are described, with exons comprising the coding sequences and introns being removed from transcripts by splicing. Alternative splicing can generate multiple protein isoforms from a single gene. Repeated sequences, including satellites, minisatellites, microsatellites, transposons, SINEs and LINEs comprise a large portion of eukaryotic genomes.
ESTs are short sequences of DNA that represent genes expressed in certain tissues or organisms. They provide a quick and inexpensive way for scientists to discover new genes and map their positions in genomes. ESTs represent a snapshot of genes expressed in a tissue at a given time. Sequencing the beginning or end of cDNA clones produces 5' and 3' ESTs, which can help identify genes and study gene expression and regulation.
Mutistep carcinogenesis refers to the process by which normal cells transform into cancerous cells through the accumulation of multiple genetic mutations over time. These mutations can be caused by environmental or inherited factors and affect genes that regulate cell growth (oncogenes) or cell cycle arrest (tumor suppressor genes). The accumulation of mutations in genes that control processes like apoptosis, cell proliferation, and DNA repair enable cells to proliferate uncontrollably and form malignant tumors.
Oncogenesis is due to uncontrolled cell growthmaryamsarwar17
Oncogenes are genes whose expression causes cells to exhibit cancer-like properties. Most oncogenes are derived from normal cellular genes called proto-oncogenes. The lecture outline discusses the identification of oncogenes in retroviruses, the mechanisms of proto-oncogene activation including mutation, translocation and amplification, the cellular locations and functions of oncogene products such as growth factors and kinases, and the roles of oncogenes like ras in the mitogen-activated protein kinase signaling pathway.
The plant nuclear genome consists of DNA organized into chromosomes within the cell nucleus. It contains both coding and regulatory sequences. The plant nuclear genome is made up of DNA, histone and non-histone proteins. DNA is packaged into nucleosomes containing histones and wrapped into chromatin. Chromatin exists in two forms - euchromatin which is loosely packaged and genetically active, and heterochromatin which is tightly packaged. Specific sequences like centromeres and telomeres aid in chromosome structure and integrity. The nuclear genome also contains both single-copy and repetitive non-coding DNA sequences.
This document provides information about physical mapping techniques used in molecular biology. It discusses that physical mapping can determine the sequence and physical distance between DNA base pairs with high accuracy. There are two main types of physical mapping: low resolution mapping, which can resolve DNA ranging from 1 base pair to several megabases, and high resolution mapping, which can resolve hundreds of kilobases to a single nucleotide. Some key techniques used for physical mapping include restriction mapping, fluorescent in situ hybridization (FISH) mapping, and sequence tagged site (STS) mapping. Restriction mapping involves cutting DNA at specific restriction sites to map fragment locations. FISH allows localization of specific DNA sequences on chromosomes using fluorescent probes. STS mapping uses short, unique
Molecular markers are DNA sequences that can be easily detected and whose inheritance can be monitored. They are based on natural polymorphisms and allow studying the inheritance of genes. Common types of molecular markers include RFLPs, RAPDs, AFLPs, SSRs, and SNPs. RFLPs use restriction enzymes to detect differences in fragment lengths. RAPDs use random primers to detect sequence polymorphisms. AFLPs selectively amplify restriction fragments to detect length differences. SSRs detect variability in simple sequence repeats. Molecular markers are useful for applications like gene mapping, phylogenetic studies, and analyzing genetic diversity.
This document discusses molecular breeding techniques using the Barnase-Barstar system for inducing male sterility in plants. It explains that the Barnase gene is cytotoxic and kills tapetum cells, preventing pollen development and resulting in transgenic male sterility. The Barstar gene provides fertility restoration. The system has been used successfully in tobacco and oilseed rape to develop hybrid seeds. Some benefits of this system include efficient fertility restoration, easy maintenance of male sterile lines, and elimination of male fertile plants from lines. However, alternative systems that are more attractive than Barnase-Barstar have also been explored.
This document discusses DNA sequencing methods. It describes the Maxam-Gilbert sequencing method developed in 1976-1977 which uses chemical modification and cleavage of DNA at specific bases, followed by electrophoresis to separate fragments by size. It also mentions the popular Sanger sequencing method. The procedure for Maxam-Gilbert sequencing involves labeling DNA, cleaving it with chemicals, running the fragments on a gel, and analyzing the results to deduce the DNA sequence. Advantages include no premature termination and ability to sequence stretches not possible with enzymatic methods, while disadvantages include use of radioactivity and toxic chemicals.
presented by HAFIZ M WASEEM
university of education LAHORE Pakistan
i am from mailsi vehari and studied in lahore
bsc in science college multan
msc from lahore
Introduction
Transcriptome analysis
Goal of functional genomics
Why we need functional genomics
Technique
1. At DNA level
2.At RNA level
3. At protein level
4. loss of function
5. functional genomic and bioinformatics
Application
Latest research and reviews
Websites of functional genomics
Conclusions
Reference
Sequence tagged sites (STSs) are short DNA sequences that can be used as genetic markers. STSs were introduced in 1989 as a way to map genes along chromosomes using PCR. They serve as landmarks on physical maps of genomes. STSs are mapped by breaking genomes into fragments, replicating the fragments in bacterial cells to create libraries, and using PCR to determine which fragments contain STSs. Different types of STS markers include microsatellites, SCARs, CAPs, and ISSRs, each of which has distinct characteristics and applications in genetic mapping, population studies, and other areas.
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
this presentation is about the molecular markers as we all know the molecular markers are the DNA sequences it can be easily detected and its inheritance is easily monitored.so the main basics of the molecular markers is the polymorphic nature so it can used as molecular markers.and this will gives you the idea about AFLP, RFLP, RAPD, SNPS,ETC.
Introduction
Backcross breeding & its types
Marker assisted breeding
Marker assisted backcross breeding (MABC)
Main strategies
Advantages over conventional breeding
Case studies
Future outlook
Conclusion
This document discusses different methods for genome sequencing and assembly, including restriction enzyme fingerprinting, marker sequences, and hybridization assays. It focuses on using marker sequences like sequence-tagged sites (STS), expressed sequence tags (ESTs), untranslated regions (UTRs), and single nucleotide polymorphisms (SNPs) to map genomes. Large-insert cloning vectors like BACs and PACs can be used with restriction enzyme fingerprinting and FPC software to assemble contigs and map genomes at a large scale. Marker sequences provide a dense set of physical markers to build accurate physical maps of genomes.
This document discusses cytoplasmic male sterility (CMS), a maternally inherited trait in plants where the plant is unable to produce functional pollen. CMS is caused by mitochondrial mutations or rearrangements that interfere with pollen development. Nuclear restorer genes can suppress CMS by interacting with the mitochondrial genes. CMS is used in hybrid seed production systems in many crops.
Gene silencing, also known as RNA interference, is a natural process in plants that evolved as a defense mechanism against viruses. Transgene silencing occurs when introduced transgenes are not expressed due to this silencing process. The first evidence of this was discovered in 1990 by R. Jorgensen in petunia plants, where both an introduced gene and endogenous gene were silenced. Gene silencing can occur at the transcriptional or post-transcriptional level through mechanisms like siRNA and microRNA production. Virus-induced gene silencing is a technique used to study gene function and develop virus-resistant plants by suppressing viral gene expression. Applications of gene silencing include developing disease-resistant crops and modifying plant traits.
Marker-assisted selection (MAS) is a plant breeding method that uses DNA markers to select for desirable traits. It allows breeders to select plants earlier in development compared to phenotypic selection. MAS has advantages like being unaffected by environment and ability to select for recessive traits, but may be more expensive initially than conventional methods. Careful analysis of costs and benefits is needed to determine if MAS is advantageous for a particular program over traditional breeding. MAS requires tightly linked markers, knowledge of marker-trait associations, and data management to be effective. A variety of MAS approaches exist like backcrossing, pyramiding, and combined MAS and phenotypic selection.
- β-glucuronidase (GUS) is a commonly used reporter gene in plant molecular biology and genetic engineering to indicate successful introduction of foreign DNA into cells.
- GUS expression can be detected through fluorometric or histochemical assays, allowing visualization of promoter activity, protein localization, and transgenic events.
- The GUS gene is fused to genes of interest, and GUS activity is used to study processes like tissue-specific expression, response to stresses, and transformation efficiency.
- While destructive, GUS is a stable and non-toxic reporter enabling versatile applications in fundamental and applied plant research.
This document describes a study that used T-DNA tagging to activate gene expression in rice. Researchers constructed vectors containing the CaMV 35S enhancer and introduced them into rice plants via Agrobacterium-mediated transformation. They generated over 13,000 transgenic lines and isolated sequences flanking the T-DNA insertions in 71 lines. They found that in 21 lines, the T-DNA inserted within 4.5kb of a gene, positioning the enhancer to potentially activate expression. Reverse transcription PCR revealed four lines where the nearby gene showed increased expression levels compared to wild-type, demonstrating the ability of T-DNA tagging to enhance gene expression.
The document discusses the C-value paradox, which is the lack of relationship between genome size and organism complexity. It provides data on the wide range of genome sizes across different taxonomic groups. Introns and exons are described, with exons comprising the coding sequences and introns being removed from transcripts by splicing. Alternative splicing can generate multiple protein isoforms from a single gene. Repeated sequences, including satellites, minisatellites, microsatellites, transposons, SINEs and LINEs comprise a large portion of eukaryotic genomes.
ESTs are short sequences of DNA that represent genes expressed in certain tissues or organisms. They provide a quick and inexpensive way for scientists to discover new genes and map their positions in genomes. ESTs represent a snapshot of genes expressed in a tissue at a given time. Sequencing the beginning or end of cDNA clones produces 5' and 3' ESTs, which can help identify genes and study gene expression and regulation.
Mutistep carcinogenesis refers to the process by which normal cells transform into cancerous cells through the accumulation of multiple genetic mutations over time. These mutations can be caused by environmental or inherited factors and affect genes that regulate cell growth (oncogenes) or cell cycle arrest (tumor suppressor genes). The accumulation of mutations in genes that control processes like apoptosis, cell proliferation, and DNA repair enable cells to proliferate uncontrollably and form malignant tumors.
Oncogenesis is due to uncontrolled cell growthmaryamsarwar17
Oncogenes are genes whose expression causes cells to exhibit cancer-like properties. Most oncogenes are derived from normal cellular genes called proto-oncogenes. The lecture outline discusses the identification of oncogenes in retroviruses, the mechanisms of proto-oncogene activation including mutation, translocation and amplification, the cellular locations and functions of oncogene products such as growth factors and kinases, and the roles of oncogenes like ras in the mitogen-activated protein kinase signaling pathway.
Oncogenic viruses can interact with host cells in ways that promote oncogenesis through both direct and indirect mechanisms. Direct mechanisms include viruses introducing oncogenes that alter cellular signaling pathways and disrupt cell cycle control. Indirect mechanisms involve evading immune responses, establishing chronic infections, and inducing chronic inflammation. Specific oncogenic viruses discussed include EBV, HPV, HBV, HCV, HTLV-1, and KSHV. Each virus employs distinct strategies to activate cancer hallmarks in host cells, with EBV and HPV expressing oncoproteins that mimic cellular signaling and proliferation factors, while HBV/HCV cause liver damage and HBV/HTLV-1 inhibit apoptosis.
This document discusses oncogenes, which are genes that can trigger cancer development. It describes several mechanisms by which normal cellular genes called proto-oncogenes can be mutated or altered to become oncogenes, including point mutations, gene amplification, chromosomal translocations, local DNA rearrangements, and insertional mutagenesis by retroviruses. Most oncogenes code for components of growth signaling pathways, such as growth factors, receptors, membrane proteins, protein kinases, transcription factors, and regulators of the cell cycle and apoptosis. Specific examples of oncogenes are provided for each of these categories.
Oncogenic viruses can cause cancer by inserting their DNA into host cells. DNA tumor viruses encode viral proteins necessary for replication that alter host cell genes and promote uncontrolled growth. RNA tumor viruses carry altered host cell genes not needed for viral replication. Key tumor suppressor genes like p53 and Rb are inactivated by viral oncoproteins from viruses such as human papillomavirus, Epstein-Barr virus, hepatitis B virus, and Merkel cell polyomavirus, leading to cellular transformation and cancer development.
This document provides an overview of murine leukemia viruses (MLVs). It discusses MLVs as both physical objects (retroviral particles) and living organisms that evolve. Key points include: MLVs are among the simplest retroviruses, encoding only Gag, Pol, and Env polyproteins; MLV infection typically does not kill host cells like HIV does; and MLVs have been useful in studying retrovirology but differ significantly from other retroviruses like HIV-1 in replication and pathogenicity. The document also describes the structure of MLV virions and genomes as well as their replication cycle.
This document discusses the mechanisms by which DNA and RNA viruses can transform cells and cause cancer. It explains that viruses can integrate their genetic material into host cell DNA, activating oncogenes and inactivating tumor suppressor genes. This disrupts the normal cell growth and division processes. Oncogenic viruses are classified based on their genetic material as either DNA tumor viruses or RNA tumor viruses, which are retroviruses. The document provides examples of specific human oncogenic viruses and discusses how viral oncogenes activate and the multi-step process of oncogenesis. Both acute and chronic transforming retroviruses are described.
This document discusses childhood Burkitt lymphoma. It begins by describing normal lymphoid tissues and cells. It then notes that Burkitt lymphoma is a highly aggressive B cell lymphoma characterized by a translocation involving the c-MYC gene. There are three clinical forms: endemic (African), sporadic, and immunodeficiency-associated. The endemic form has the highest incidence in Africa and peaks in children ages 4-7 years. Diagnosis involves identifying a rapidly growing tumor with a "starry-sky" appearance under microscopy. Key investigations include blood work, imaging scans, and tissue biopsies.
Genetics plays a key role in cancer development. Cancer results from mutations in proto-oncogenes, tumor suppressor genes, miRNA genes, and mutator genes. Proto-oncogenes promote cell growth but become oncogenes when mutated. Tumor suppressor genes normally inhibit cell growth but are inactivated by mutations in cancer. The cell cycle is tightly regulated by checkpoints but deregulation can lead to uncontrolled cell division in cancer. Cancers can be caused by inherited genetic mutations or environmental exposures like radiation and carcinogens. The development of cancer typically involves multiple genetic alterations accumulating over time.
Viruses are complexes consisting of protein and an RNA or DNA genome that lack cellular structure and independent metabolism. They replicate solely by exploiting living cells based on the information in their genome. Viruses come in a variety of sizes and structures, and can have DNA or RNA genomes. They reproduce by first binding to and entering a host cell, then exploiting the cell's machinery to produce new viral components and assemble new virus particles, which then burst out to infect other cells. Viruses are classified based on properties like their genome, capsid symmetry, presence of an envelope, and more. While they cannot be treated with antibiotics, some antiviral drugs can inhibit specific stages of the viral replication process.
here i discussed some human oncogenic viruses , their epidemeology, life cycle, treatment, prevention and control. . oncogenic viruses are cancer causing viruses.
The document summarizes evasion mechanisms used by viruses to avoid the host immune system. It discusses two main mechanisms used by influenza viruses: antigenic drift which involves point mutations in surface proteins, and antigenic shift which involves reassortment of RNA segments between animal and human influenza viruses. It also describes how herpes simplex viruses prevent the transport of viral peptides to the ER through the protein ICP-47, inhibiting antigen presentation through MHC class I molecules on infected cells.
This document discusses various aspects of carcinogenesis. It begins by describing the key properties of cancer cells such as uncontrolled growth, invasion, and metastasis. It then discusses various causes of cancer including physical, chemical, and biological agents. Radiation, chemicals, and viruses can all act as carcinogens. The document goes on to describe how carcinogens interact with and damage DNA. It discusses initiation and promotion in carcinogenesis and the roles of oncogenes and tumor suppressor genes such as p53. The document also covers topics like telomerase, metastasis, and the tendency of tumors to progress in malignancy.
Viral oncogenesis can be caused by retroviruses, DNA viruses, or RNA viruses. Retroviruses may contain cellular oncogenes called v-onc genes. Some retroviruses induce cancer by inserting near cellular proto-oncogenes (c-onc genes), deregulating their expression. c-onc genes encode proteins involved in growth control and differentiation. Their mutation or overexpression can lead to cancer. Both DNA and RNA tumor viruses can integrate into host genomes and induce transformation by expressing early genes. Human viruses linked to cancer include EBV, HBV, HCV, HPV, and HIV. HIV causes AIDS by depleting CD4+ T cells, leaving the immune system vulnerable to opportunistic infections.
RETROVIRUS MEDIATED GENE TRANSFER AND EXPRESSION CLONINGSrishtiRoy10
- The retroviral virion is a spherical particle 80-100 nm in diameter composed of a lipid bilayer envelope containing glycoproteins and a capsid containing two copies of the viral RNA genome and enzymes.
- Retroviruses replicate by reverse transcribing their RNA genome into DNA which is then integrated into the host cell genome by an integrase enzyme to become a provirus, allowing transcription of viral genes.
- Retrovirus mediated gene transfer involves the virus producing a DNA copy of its genome using reverse transcriptase, with the DNA then integrating randomly into the host cell genome, allowing investigation of gene function.
A detailed description of HIV covering virology, morphology, pathogenesis, clinical stages and manifestations, laboratory diagnosis, and diagnostic strategy, and therapeutic options and prevention.
This document provides an overview of Hepatitis C. It begins with an introduction stating that over 71 million people worldwide are chronically infected with HCV. It then covers the virology of HCV including its structure, genome, replication cycle, genotypes/quasispecies. The epidemiology section discusses the global prevalence and incidence. Pathogenesis outlines how HCV evades the immune system to cause chronic infection. Clinical features are separated into acute hepatitis C and chronic hepatitis C. Extrahepatic manifestations associated with HCV are also summarized.
share - Lions, tigers, AI and health misinformation, oh my!.pptxTina Purnat
• Pitfalls and pivots needed to use AI effectively in public health
• Evidence-based strategies to address health misinformation effectively
• Building trust with communities online and offline
• Equipping health professionals to address questions, concerns and health misinformation
• Assessing risk and mitigating harm from adverse health narratives in communities, health workforce and health system
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ABDOMINAL TRAUMA in pediatrics part one.drhasanrajab
Abdominal trauma in pediatrics refers to injuries or damage to the abdominal organs in children. It can occur due to various causes such as falls, motor vehicle accidents, sports-related injuries, and physical abuse. Children are more vulnerable to abdominal trauma due to their unique anatomical and physiological characteristics. Signs and symptoms include abdominal pain, tenderness, distension, vomiting, and signs of shock. Diagnosis involves physical examination, imaging studies, and laboratory tests. Management depends on the severity and may involve conservative treatment or surgical intervention. Prevention is crucial in reducing the incidence of abdominal trauma in children.
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2. MUTAGENESIS :
It is a process by which the genetic information of an organism is
changed in a stable manner resulting in a mutation.
May occur spontaneously in nature or as a result of exposure to
mutagens or achieved by experimental laboratory procedures.
3. HISTORY OF MUTAGENESIS
DNA may be modified, either naturally or artificially, by a number of
physical, chemical and biological agents, resulting in mutations.
In 1927, Herman Muller, first demonstrated that mutation with observable
changes in the chromosomes can be caused by irradiating fruit flies with X-
ray.
Lewis Stadler also showed the mutational effect of X-ray on barley in 1928
and ultraviolet (UV) radiation on maize in 1936.
In 1978, Michael Smith discovered site-directed mutagenesis by using
oligonucleotides in a primer extension method with DNA polymerase.
5. INSERTIONAL MUTAGENESIS
Insertional mutagenesis is the phenomenon by which an exogeneous
DNA sequence integrates within the genome of a host organism.
This event can result in the deregulation of genes in the neighborhood
of the insertion site and can potentially cause a perturbation of cellular
phenotype.
It has been widely exploited for forward genetics screening aimed at
identifying novel cancer gene.
6. REQUISITES FOR INSERTIONAL MUTAGENESIS
I. Proper oncogenic agent that efficiently integrates in a random
or semi-random fashion into the host genome
II. An animal model that is permissive to tumor formation
III. Efficient technologies to perform retrieval of integration sites
IV.Bioinformatic and static tools to map integrations onto the host
genome and identifying targeted sites, defined as CIS (CIS –
Common insertion site) that are locations hosting candidate
cancer genes.
7. MECHANISM OF (INSERTIONAL MUTAGENESIS)
Integrating vectors can induce cancer by mutating host genes in
a number of different ways and they can enhance transcription or
translational levels of oncogenes, generated chimeric or
truncated transcripts or inactivate TSG expression.
Basically, there are three types of insertions –
(i) Enhancer insertions
(ii) Promoter insertions
(iii) intragenic insertions
8. ENHANCER INSERTION
It is a common mechanism of mutagenesis (virus) and results in the
up-regulation of endogenous gene expression.
As, regulatory elements may be distal to a gene, enhancer
insertions can be located some distance from the proximal promoter
and may affect the activity of elements via chromatin loops, thus
complicating the identification of the affected genes.
It may be independent from their orientation, but are classically
upstream of a mutated gene in the antisense orientation or
downstream in the sense orientation, especially for retrovirus.
9. PROMOTER INSERTION
It occurs when the insertional mutagen integrates in the sense
orientation in or close to the proximal promoter region of an
endogenous gene and uncoupling the cellular gene from its
promoter and placing it under the control of elements found
within the insertional mutagen.
RNA polymerase transcribes an RNA starting from the promoter
of the insertional mutagen that reads through the introns and
exons of the host gene.
The integrating vector contains splicing signals that induce the
joining of the first vector – coded exon to those of the host gene
and thus, can result in the translation of high levels of chimeric
transcripts.
10. INTRAGENIC INSERTION
Intragenic insertions can interfere with the splicing of genes into
which they integrate, as many of insertional mutagens contain poly A
signals (engineered/endogenous), that may elicit premature
termination of gene transcription.
Truncation of the transcripts may have different consequences on the
resulting protein and result in the expression of altered or neomorphic
alleles.
viral insertion in the 3’UTR region of a gene may remove mRNA-
destabilizing motifs such as AUUUA hairpins or miRNA target
sequences and thus, resulting in increased levels of truncated but wild-
11. CONTINUED….
By this mechanism, insertional mutagens may induce the
formation of a 3’ truncated mRNA by removing from the transcript
protein coding exons.
Alternatively, transcription may start from the integrated
promoter, 5’ truncated mRNA is transcribed and hence, resulting
in C-terminal or N-terminal truncated proteins may possess
oncogenic properties and induce tumorigenesis.
Using the same mechanism, vector insertions can inactivate a
gene: landing within a gene may result either in an mRNA
encoding an inactive or unstable protein or in aberrant splicing
which abrogate the gene function.
12. INTEGRATING AGENTS
The integrating agents that are efficiently used so far to identify
new cancer genes by insertional mutagenesis are retroviruses and
transposable elements.
RETROVIRUSES
these are large family of enveloped RNA viruses found in all
vertebrates
the genome is a homodimer of linear, positive sense, ssRNA of 7-
11Kb, surrounded by a cone shaped protein core.
In the retroviral life-cycle, the genetic information goes from RNA to
DNA and exists in two different forms; as genomic DNA when inside
the viral particle and as proviral double-stranded DNA when
integrated in the host genome.
13. CONTINUED….
Both the ends of the genome contain terminal noncoding sequences, the so-called
long terminal repeats (LTR), composed of 5’ and 3’ unique sequences (U5 and U3
regions) and of two direct repeats (R) where the transcription start site(TSS) and the
polyadenylation signals (polyA) are located.
Upon interaction between envelope glycoproteins and cellular receptors, fusion of the
virus and host cell plasma membrane occurs, and the viral capsid containing the RNA
genome enters the cell.
The viral RNA genome is released in the cytoplasm and subsequently retrotranscribed
into a double-stranded proviral DNA.
DNA is associated with viral proteins, and translocates to the nucleus, where the
integrase mediates integration of the provirus into the host cell genome.
Oncogenic retroviruses have been classified in two groups, acute- and slow-
transforming retroviruses
acute transforming retroviruses – it induces the formation of polyclonal cancers
by highly expressing virus-encoded oncogenes with short latency periods (2-3 weeks) .
14. Structure of the genome
and replication life cycle
of
retrovirus
15. RETROVIRUSES AS INSERTIONAL
MUTAGENS
From many years, insertional mutagenesis have used two types of slow
transforming ssRNA retroviruses: Moloney murine leukemia virus
(MoMLV , ɤ retrovirus ) and mouse mammary tumor virus (MMTV, α
retrovirus).
These viruses cause lymphomas and mammary tumors in mice.
Single genomic integration is not enough to promote tumorigenesis
and multiple rounds of proviral insertions within the same genome are
required for the development of cancer.
By cloning proviral insertion sites from tumor cells it is possible to
identify candidate genes that have contributed to the development of
16. MURINE LEUKEMIA VIRUSES IN HEMATOPOIETIC MALIGNANCIES
Moloney murine leukemia virus (MoMLV) is the prototypical murine
leukemia virus whose integration have been extensively studied for the
discovery of cancer genes in mice.
The U3 promoter of MMLV recruits the basal transcriptional
machinery as it contains a TATA box and GC-rich sequences.
The enhancer element within the virus has binding sites for B and T-
cell–specific transcription factors, such as ETS, NF1, RUNX, and MYB.
MoMLV mainly induces T and B cell leukemia/lymphoma in mice.
17. CONTINUED….
By analysing MoMLV integrations in murine tumors, several oncogenes have
been identified, including c-Myc, Pim1, Pim2, Pvt 1.
Many studies have created recombinant MLVs by substituting LTRs from
other viruses and have tested them in mice.
In, 2012 , Starkey et al., replaced the MoMLV LTRs with those of feline
leukemia virus and named it MoFe2-MuLV, mainly induced T-cell
lymphoma.
Interestingly, analysis of MoFe2-MuLV integrations showed that this virus
did not target the same site as MoMLV and indeed led to the identification of
new cancer genes; Mf8t, Jundm2, Ahi1, Rras2.
18. CONTINUED….
The SL3-3 MLV strain typically induces T-cell lymphomas, but a range of
LTR mutations, such as mutation of RUNX-binding sites, extends cancer
latency and skews tumor formation causing myeloid, B-lymphoid, and
erythroid tumors.
MLVs are specific for different hematopoietic compartments, for
instance the erythroleukemia-inducing Friend virus is more active in
erythroid cells, whereas the Graffi-1.4 virus induces mainly acute myeloid
leukemia (AML).
Using MMLV in transgenic E μ-Myc mice induced the development of
B-cell lymphoma and allowed the identification of additional cancer
19. CONTINUED….
• Instead of injecting a viral supernatant in newborn mice, identified CIS in tumors
derived from the recombinant inbred strains, BXH2 and AKXD.
• In BXH2 mice, the MLV is not inherited through the germline, rather is horizontally
transmitted via transplacental infection of implanted embryos.
• In the AKXD strains, the MLV is inherited through the germline, but then somatic
activation of additional recombinant viruses occurs.
• Therefore, the virus starts to replicate and integrates into the genome during early
gestation with mutagenesis continuing throughout the life of the animal causing a life-
long viremia.
• Indeed, studying and comparing the CIS retrieved from these mice allowed identifying
lineage-specific cancer genes involved in hematopoietic malignancies that are also
relevant in human tumors.
20. General outline of the work flow for cancer gene discovery using insertional mutagens
21. MMTV IN MAMMARY CANCER
Mouse mammary tumor virus (MMTV) is a retrovirus with a similar tumorigenic
behaviour as MoMLV but with a specific tropism for mammary cells.
Since carcinomas represent the most frequent human malignancies, the ability
of MMTV to hit the epithelial compartment is valuable and has resulted in the
identification of many clinically relevant cancer genes.
The MMTV life cycle usually begins with the ingestion of infected milk by pups
from their viremic mother and after a few days, the virus infects B cells in
lymphoid tissue of the gut, for example, Peyer's patches.
Thereby, stimulating a T-cell antigen response, a pool of B and TMMTV-
infected cells is generated as a reservoir of virus, from which MMTV spreads
into different tissues ultimately resulting in transformation of mammary
epithelial cells.
22. CONTINUED…..
Since 1982, MMTV has been used for forward genetic studies when
Nusse and Varmus identified Wnt1 as a frequent insertion target.
Different studies conducted with MMTV-induced tumors led to the
identification of 12 candidate cancer genes, mainly belonging to Wnt
and Fgf pathways.
But recently, Theodorou et al., performed the first large scale MMTV
insertional mutagenesis screening in the mouse mammary gland and
160 mammary tumors were screened and identified 33 CIS (among
which 21 were previously unknown MMTV targeted genes).
23. TRANSPOSON MEDIATED INSERTIONAL
MUTAGENESIS
Transposons are DNA sequences that can move from one location on
the genome to another.
There are two general classes of transposons: retrotransposons and
DNA transposons.
RETROTRANSPOSONS
• They move by a “copy and paste” process (replicative transposition),
transposing via an RNA intermediates, which is then converted to DNA by
retrotranscription, which then inserts in new locations in the genome.
• But the low integration efficiency, the integration of incomplete
retrotranscribed elements and the concomitant induction of
chromosomal aberrations are still limiting their applicability to cancer
24. DNA TRANSPOSONS
DNA transposons are a class of "cut and paste" transposons that rely
on a transposase enzyme, which recognizes specific DNA sequences
and "cuts" the DNA between them.
The excised DNA is then reintegrated at another site in the genome.
DNA transposon-mediated insertional mutagenesis screenings have
been developed in the last decade and allow the generation of tumors
in a wider spectrum of tissues than the ones that are accessible using
retroviruses.
25. SLEEPING BEAUTY SYSTEM
• DNA transposons are actively mobile only in plants and invertebrates, the
Sleeping Beauty transposon system was generated by reverse engineering in
1997, based on sequence comparison between multiple salmonid species of
nonfunctional transposase genes of the Tc1/mariner family that had
accumulated inactivating mutations.
• The Sleeping Beauty system is composed of two elements: a transposase,
that is, the enzyme responsible for mobilization, and the transposon, that is,
the actual mobilized sequence of DNA, which is flanked by the binding sites
for the Sleeping Beauty transposase, the so-called inverted repeats/direct
repeats.
• Transposition occurs when the transposase binds two sites in each inverted
repeat/direct repeat, and the closer the inverted repeats/direct repeats, the
26. CONTINUED….
When the transposase excises a transposon, it leaves behind a three-
base footprint.
The transposon can then reinsert at any location in the genome
where a TA dinucleotide is present (there are more than 300 million
TA sites in the genome) and during integration, the TA is duplicated.
The cargo of the transposon (the segment contained between the two
inverted repeats/direct repeats) can be any sequence of choice, but
transposition efficiency decreases with increased cargo sizes .
But, by introducing point mutations in the transposase gene, it has
been possible to significantly increase the level of mobilization.
28. CONTINUED….
Transposition may be controlled by separating the transposase from the
transposon, thus establishing a nonautonomous bipartite transposon
system.
In such a bipartite system, the transposon can be mobilized only when
the transposase protein is expressed.
This may be achieved by generating two transgenic mouse strains: the
first is called the "jumpstarter" strain expressing the transposase gene;
the second is the "mutator" strain carrying the nonautonomous
transposon.
29. PIGGYBAC SYSTEM FOR INSERTIONAL MUTAGENESIS
AND CANCER GENE DISCOVERY
Other transposable elements have been developed and exploited such as Minos (from
Drosophila hydei) and Tol2 (from Oryzias latipes, the Medaka fish, Zebrafish).
To date, the only transposon that has been efficiently exploited for cancer gene
discovery as an alternative to Sleeping Beauty is the PiggyBac (PB) system.
PB is a DNA transposon from the cabbage looper moth Trichoplusia ni that has been
developed to be active in mammalian cells, including mice.
30. CONTINUED….
PIGGYBAC SYSTEM SLEEPING BEAUTY
SYSTEM
Can efficiently mobilise large cargos it can mobilise smaller cargos
they integrate a TTAA tetranucleotide
sequence
they integrate TA dinucleotide sequence
Doesn’t leave any footprints Leaves three base pair footprints
Damages the neighbouring genome near
the mobilisation site
Doesn’t disrupt the genome nearby
31. CONTINUED….
In 2010, in the Science, they published PB insertional mutagenesis to identify
cancer genes in mice and deployed a bifunctional transposon containing 5’ and 3’
consensus sequences for both the Sleeping Beauty and PB transposases.
The design of the integrating cassettes used was similar to T2/Onc transposon
because they contained a promoter, splice donor, splice acceptor and polyA sites to
generate gain-of-function and loss-of-function mutations.
Three different types of transposons were developed, each containing a different
enhancer/promoter: MSCV (Murine stem cell virus), CAG, or the phosphoglycerate
kinase (PGK) promoter.
Nineteen different transposon lines were produced from these three different transposons
each containing a different number of copies resident at different chromosomal loci.
33. CONTINUED….
• Fourteen of these lines were crossed with
mice that constitutively express PB
transposase from the Rosa26 locus.
• But, in case of they observed a high rate of
embryonic lethality in mouse strains that
contain a high copy number of
transposons. However, the strains with
intermediate to low copy number,
transposon arrays tumors formed.
• The tumor latency and the tumor type
were found to be profoundly affected by
the type of transposon.
34. CONTINUED….
Mobilization of the MSCV transposon resulted in more than 90%
hematopoietic tumors.
Conversely, the CAG transposon mainly induced solid tumors, including
sarcomas and various carcinomas with poor differentiation, and in some
cases metastasis was observed.
Mice carrying mobilized PGK transposons developed hematopoietic and solid
tumors, with several mice bearing both types of malignancies.
Remarkably, 42% of the candidate cancer genes identified by PB insertional
mutagenesis were not significantly hit in the previous studies conducted
using retroviruses or Sleeping Beauty transposons, suggesting that PB
targets a unique spectrum of loci.
35. ADVANTAGES OF INSERTIONAL MUTAGENESIS
SYSTEM
• The recent advance in sequencing
technologies has boosted the analysis of
cancer genomes at different levels,
including identification of point
mutations, chromosomal aberrations, and
epimutations
• High-throughput screening with cDNA
libraries or short hairpin RNA (shRNA)
libraries have also been exploited to
identify oncogenes and TSGs
LIMITATIONS
- These approaches require hundreds or
thousands of tumors to be analyzed to
implicate in cell transformation of those
genes that are less frequently mutated
thus making data from functional
approaches
LIMITATIONS
- cDNA libraries may not express a gene
product at an appropriate level to induce
tumorigenesis, whereas shRNAs are
notorious for off target effects and
incomplete silencing.
38. INTRODUCTION
Traditional vertebrate model system such as mouse have now been complemented by
other organisms; rat, diploid frog (Xenopus tropicalis) and Zebrafish (Danio rerio).
The insertional mutagenesis approach has now being deployed as an appropriate
methodology for genome modification in other model vertebrates that lack ES cells,
such as rat and pig.
Advantages of the zebrafish include its external fertilization, high fecundity, rapid
development, production of optically clear embryos, and relatively short generation
time for a vertebrate.
In addition to the high degree of genetic conservation reflected in the developmental
gene pathways and regulatory mechanism, contribute to its emergence as a model for
obtaining insights into fundamental human physiology.
39. CONTINUED….
They established forward genetic tools for the zebrafish include chemical
(N-ethyl-N-nitrosourea [ENU]; and insertional (retroviral) mutagens.
Here, ENU produced random point mutations in the germline and these
single base pair changes resulted in a high frequency of mutant phenotypes.
In spite of the high efficiency in generation of point mutations, the major
limitation in this approach, was identification of genes whose mutations are
responsible for the particular phenotype.
An alternative approach is insertional mutagenesis, in which an exogenous
DNA serves as a mutagen and also functions as a molecular tag for
identifying the gene whose disruption causes the phenotype.
41. RETROVIRAL INSERTIONAL MUTAGEN IN
ZEBRAFISH
• The most extensively studied insertional mutagen to date in zebrafish is the
pseudotyped retrovirus, composed of a genome based on the Moloney murine
leukemia virus and the envelope glycoprotein of the vesicular stomatitis virus .
• They have injected this retrovirus into 1,000-cell to 2,000-cell stage zebrafish
embryos results in chimeric embryos in which different cells have integrations
of the viral sequences in different random sites in the genome.
• By passing these insertions through the germline and inbreeding them, they
have identified more than 500 mutations and about 350 loci; the insertional
nature of the mutagen facilitated the rapid molecular characterization of the
genetic loci, with 335 clones.
42. CONTINUED….
Retroviruses appear to cause mutations in Zebrafish by several major mechanisms,
including exon disruption and gene silencing caused by insertion into an intron.
Nearly 30% of mutagenic insertions recovered from a large scale mutagenesis
screening were in exons, basically in the 5’ UTR and such insertions lead to a complete
loss of wild-type gene product.
And rest most of the other 70% of mutagenic insertions were in introns, as because
the MoMLV prefer to get inserted near the 5’ end of genes and generally these
insertions were in the first intron.
Hence, these insertions usually result in the reduction or complete abrogation of
endogenous RNA Expression.
43. CONTINUED….
Intronic insertions can also lead to aberrant splicing, resulting in skipped exons
and either frameshift mutations or internally truncated gene products.
One major challenge to the field has been the inability to develop a similarly
mutagenic, high-titer retrovirus with robust expression.
44. TRANSPOSON BASED INSERTIONAL MUTAGENS IN
ZEBRAFISH
They have employed a 5’ gene trap vectors in zebrafish and this trap was used to
increase the mutagenicity of retroviral vector.
This vector utilized a splice-in and splice-out vector method, with intronic
insertion in the correct orientation inducing a frameshift and probably resulted,
either a truncated protein or a loss of gene product due to nonsense-mediated
mRNA decay.
The 5’ gene trap contained, a splice acceptor and the green fluorescent protein
(GFP) gene was used in a Tol2 based transposon insertional study in Zebrafish.
Integration of the gene trap in the proper orientation and reading frame, resulted in
GFP expression in temporally and spatially restricted pattern.
In this study, thirty six trapped lines were homozygosed with no visible phenotypes,
but only about 5% of zebrafish genes had shown embryonic phenotypes when
46. CONTINUED….
They concluded that this type of gene traps may not reliably mutate
gene.
Because many insertions in introns (the type of insertion required to
activate this trap) can abrogate or severely reduce gene expression.
It may be that many such insertions were not detected as trap events
because the GFP reporter cannot be visualized in the absence of
expression of the endogenous gene.
47. CONTINUED….
A combined 5’-3’ gene trap (‘gene breaking’) vector was developed to trap genes in
zebrafish.
They have included a 5’ transcriptional terminator cassette to mutate the
gene in concert with 3’ gene trapping as an alternative strategy to select for
intragenic vector integrations.
Although the employment of a transcriptional terminator in the gene breaking
trap vector allows suppression of splicing around the trapping vector, the
trapped gene expression domain cannot readily be identified using this basic
approach.
Alternative approaches to add this feature to gene breaking transposons are
underway.
48. CONCLUSION
Importantly, the recent studies with PB transposons clearly showed that using novel
tools allowed the identification of novel culprits of cell transformation, which were
elusive in the previous screening.
Moreover, a-retroviral vectors have been recently developed for gene therapy
Applications.
Insertional mutagenesis plays also a role in human disease and recent studies have
shown that spontaneous retrotransposon integration has a role in the pathogenesis of
different human tumors.
The continuous development of new insertional mutagenesis tools, together with the
improvement of sequencing technologies for the retrieval of integration sites, will
continuously boost these forward genetics screenings that promise to significantly