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1. The study screened signaling pathways to identify regulators of quiescence in the Drosophila testis stem cell niche. 2. Knocking down Wg in the hub produced one mitotic hub cell out of 27 testes, indicating Wg may regulate hub quiescence. 3. Most pathways tested, including JAK/STAT and EGFR, did not change hub quiescence when components were knocked down.
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Microbial Genetics: Transformation, Transduction, Conjugation, Plasmids, Tran...
This document discusses various techniques in microbial genetics including transformation, transduction, conjugation, plasmids, and transposons. Transformation involves the uptake of genetic material like DNA by bacterial cells. Transduction occurs when viruses called bacteriophages transfer genetic material between bacteria. Conjugation is the transfer of genetic material like plasmids through direct contact between bacteria. Plasmids are small circular DNA molecules that are distinct from chromosomal DNA and often provide genetic advantages to bacteria. Transposons are genetic elements that can move to different locations in a genome and contribute to the spread of traits like antibiotic resistance.
Bacterial conjugation and its application
Bacterial cells rise their level of genetic diversity and overcome their lack of sexuality by horizontal DNA transfer.
Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells.
Conjugation
Conjugation is the transfer of genetic material between bacterial cells that are in direct contact. Joshua Lederberg and Edward Tatum discovered conjugation in 1946 through an experiment where they mixed two auxotrophic E. coli strains that could complement each other's mutations. Bernard Davis provided evidence in 1950 that direct cell contact was required by showing that genetic transfer did not occur when the two strains were separated by a filter. During conjugation, a pilus forms to attach the donor and recipient cells and allows single-stranded DNA to pass between them to make both cells viable donors that can now transfer the genetic material.
Tessa Poster for IP
1. 22Rv1 prostate cancer cells were treated with enzalutamide to generate resistant cells (22Rv1-R).
2. Microscopy showed no morphological differences between 22Rv1 and 22Rv1-R cells. Western blot analysis revealed higher MDR1 expression in 22Rv1-R cells.
3. MTT assay demonstrated 22Rv1-R cell viability remained stable in increasing enzalutamide concentrations, indicating resistance, while 22Rv1 cell viability decreased.
Gene transfer in bacteria
This document provides an overview of gene transfer in bacteria through three main methods: conjugation, transformation, and transduction. Conjugation involves the transfer of genetic material between bacteria via cell-to-cell contact through sex pili. Transformation refers to the uptake of naked DNA by competent bacterial cells. Transduction is the transfer of DNA from one bacterium to another via bacteriophage. Each method is described in 1-2 paragraphs detailing its history of discovery and basic mechanisms.
Genetic engineering in animal cells
This document outlines methods for creating transgenic animals. It begins with definitions and historical background, then describes the general strategy which involves isolating a gene of interest, generating a DNA construct, injecting the construct into embryos, and analyzing expression in offspring. Three main methods are discussed: DNA microinjection, retrovirus-mediated gene transfer, and embryonic stem cell-mediated gene transfer. DNA microinjection involves directly injecting DNA into the nucleus of cells, but results in transgenic progeny in less than 5% of cases. Retroviral vectors can be used to introduce genes. Embryonic stem cells are modified then injected into blastocysts. The document also covers getting embryonic cells, selecting transfected cells using genetic markers,
GENE KNOCKOUT
A gene knockout is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, gene knockout can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals.
Gene therapy : Types, Gene transfer methods vectors for gene therapy approach...
Gene therapy: Types of Gene therapy Gene transfer methods vectors for gene therapy approaches applications advantages and disadvantages. Gene therapy based drugs. Ethical considerations.
Recommended
Microbial Genetics: Transformation, Transduction, Conjugation, Plasmids, Tran...
This document discusses various techniques in microbial genetics including transformation, transduction, conjugation, plasmids, and transposons. Transformation involves the uptake of genetic material like DNA by bacterial cells. Transduction occurs when viruses called bacteriophages transfer genetic material between bacteria. Conjugation is the transfer of genetic material like plasmids through direct contact between bacteria. Plasmids are small circular DNA molecules that are distinct from chromosomal DNA and often provide genetic advantages to bacteria. Transposons are genetic elements that can move to different locations in a genome and contribute to the spread of traits like antibiotic resistance.
Bacterial conjugation and its application
Bacterial cells rise their level of genetic diversity and overcome their lack of sexuality by horizontal DNA transfer.
Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells.
Conjugation
Conjugation is the transfer of genetic material between bacterial cells that are in direct contact. Joshua Lederberg and Edward Tatum discovered conjugation in 1946 through an experiment where they mixed two auxotrophic E. coli strains that could complement each other's mutations. Bernard Davis provided evidence in 1950 that direct cell contact was required by showing that genetic transfer did not occur when the two strains were separated by a filter. During conjugation, a pilus forms to attach the donor and recipient cells and allows single-stranded DNA to pass between them to make both cells viable donors that can now transfer the genetic material.
Tessa Poster for IP
1. 22Rv1 prostate cancer cells were treated with enzalutamide to generate resistant cells (22Rv1-R).
2. Microscopy showed no morphological differences between 22Rv1 and 22Rv1-R cells. Western blot analysis revealed higher MDR1 expression in 22Rv1-R cells.
3. MTT assay demonstrated 22Rv1-R cell viability remained stable in increasing enzalutamide concentrations, indicating resistance, while 22Rv1 cell viability decreased.
Gene transfer in bacteria
This document provides an overview of gene transfer in bacteria through three main methods: conjugation, transformation, and transduction. Conjugation involves the transfer of genetic material between bacteria via cell-to-cell contact through sex pili. Transformation refers to the uptake of naked DNA by competent bacterial cells. Transduction is the transfer of DNA from one bacterium to another via bacteriophage. Each method is described in 1-2 paragraphs detailing its history of discovery and basic mechanisms.
Genetic engineering in animal cells
This document outlines methods for creating transgenic animals. It begins with definitions and historical background, then describes the general strategy which involves isolating a gene of interest, generating a DNA construct, injecting the construct into embryos, and analyzing expression in offspring. Three main methods are discussed: DNA microinjection, retrovirus-mediated gene transfer, and embryonic stem cell-mediated gene transfer. DNA microinjection involves directly injecting DNA into the nucleus of cells, but results in transgenic progeny in less than 5% of cases. Retroviral vectors can be used to introduce genes. Embryonic stem cells are modified then injected into blastocysts. The document also covers getting embryonic cells, selecting transfected cells using genetic markers,
GENE KNOCKOUT
A gene knockout is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, gene knockout can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals.
Gene therapy : Types, Gene transfer methods vectors for gene therapy approach...
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GENE THERAPY AND ITS APPLICATION
GENE THERAPY: TYPES, METHODS, FACTORS AND STANDARDS AND ITS APPLICATION IN HEALTHCARE FIELD
INVIVO THERAPY AND EXVIVO THERAPY
CHEMICAL AND PHYSICAL METHODS TO CARRY ON GENE THERAPY
DEFECTIVE GENE IDENTIFICATION IN GENE THERAPY AND TREATMENT OF GENETICALLY AFFECTED GENE BY GENE THERAPY
Transduction and transformation
Transduction is the process by which DNA is transferred between bacteria via a virus. It involves a bacteriophage infecting a bacterial cell and using the cell's machinery to produce new virus particles, which may incorporate small fragments of bacterial DNA into new viral genomes. This allows genetic material to be transferred from one bacterium to another without direct contact when the new viruses infect other bacteria. Transduction differs from transformation in that the transferred DNA is protected from DNase inside the viral particle. It is a molecular biology tool for introducing foreign DNA into host genomes.
MFF Poster-GRBaV-AC.pptx
1. Researchers at Cornell University studied the transcriptome of Grapevine Red Blotch-associated Virus (GRBaV) which causes Grapevine Red Blotch Disease. 2. They successfully extracted RNA from infected grapevine leaves and detected several discrete viral RNA transcripts between 100-3,200 nucleotides in size using northern blot hybridization. 3. Preliminary 5’-RACE sequencing results aim to identify the 5’-ends of viral mRNAs to better understand GRBaV transcription strategies.
Dna transfer
This document discusses various methods of transfection, which is defined as the introduction of foreign DNA into eukaryotic cells. It describes transfection methods such as calcium phosphate transfection, liposome-mediated transfection, retroviral transfection, and electroporation. It provides details on how each method works and compares their strengths and weaknesses. Common transfection methods like calcium phosphate and liposomes are simple but have low efficiency, while retroviral transfection can generate stable cell lines but has limitations on DNA size. Electroporation is fast and applicable to many cell types.
polymorphism
Genetic polymorphisms are normal variations that occur in more than 1% of the population. Single nucleotide polymorphisms are the most common type, involving a change in a single DNA base pair. Polymorphisms can occur in coding and regulatory regions, and may affect protein function or expression levels. The interleukin-1 gene cluster contains polymorphisms that have been associated with increased severity of periodontitis, where individuals with certain IL-1 genotypes experience more rapid bone and tissue loss around teeth. Understanding genetic predispositions can help with early detection and prevention of more serious disease.
Genetic polymorphism and It's Applications
Genetic polymorphism
Genetic polymorphism is the inheritance of a trait controlled by a single genetic locus with two alleles, in which the least common allele has a frequency of about 1% or greater. Genetic polymorphism is a difference in DNA sequence among individuals, groups, or populations.
Types of polymorphisms
Protein/enzyme polymorphisms
In the early days of human genetics, majority of polymorphisms were those associated with proteins and enzymes. To detect the polymorphism and a person’s genotype, one performed assays for the gene product, i.e., the protein or enzyme produced by the genetic blueprint.
DNA polymorphisms
The large class of polymorphisms are those that detect Slight variations at the level of DNA nucleotides.
Single nucleotide polymorphisms
A single nucleotide polymorphism or SNP is a sequence of DNA on which humans vary by one and only one nucleotide . Because humans differ by one nucleotide per every thousand or so nucleotides, there are millions of SNPs scattered throughout the human genome.
Tandem repeat polymorphisms
A tandem repeat polymorphism consists of a series of nucleotides that are repeated in tandem (i.e., one time after another). The polymorphism consists of the number of repeats.
Restriction Fragment Length Polymorphism (RFLP)
Restriction Fragment Length Polymorphism (RFLP) is a type in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA. If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme.
Applications of Genetic Polymorphism
The study of polymorphism has many uses in medicine, biological research, and law enforcement. Genetic diseases may be caused by a specific polymorphism. Scientists can look for these polymorphisms to determine if a person will develop the disease, or risks passing it on to his or her children.
Gene Therapy, Applications and Recent advances
This document discusses gene therapy and is presented by Urvashi Shakarwal. It defines gene therapy as the insertion of genes into an individual's cells to treat a disease. It describes the different approaches to gene therapy, including viral and non-viral vectors. Viral vectors commonly used are retroviruses, adenoviruses, and herpes simplex viruses. Non-viral methods include electroporation, microinjection, gene guns, and calcium phosphate or liposome transfection. The document outlines several applications of gene therapy, such as for chronic granulomatosis disorder, hemophilia, and inherited blindness.
Genetic polymorphism
The document discusses genetic polymorphisms in Plasmodium falciparum, the parasite that causes malaria. It defines key terms like locus, allele, and genome. It then describes different types of genetic polymorphisms like single nucleotide polymorphisms (SNPs), insertions and deletions (INDELs), and short tandem repeats (STRs). The document focuses on polymorphisms related to drug resistance in P. falciparum, discussing genes associated with resistance to chloroquine (pfcrt) and other antimalarial drugs, along with specific mutations in those genes linked to resistance.
Gene therapy
Gene therapy , Nucleic acids , GENES , ADA-SCID(Severe combined immunodeficiency) , APPROACHES IN GENE THERAPY : Types of gene therapy , somatic gene therapy , germ line gene therapy , Gene modification , gene replacement , gene correction , gene augmentation , Gene transfer methods , viral gene transfer (biological) , non viral gene transfer : physical method ,chemical method , retrovirus vector system , adenovirus vector system , Adeno associated virus vector , Herpex simplex virus vector , Electroporation , Microinjection , Gene Gun , Calcium phosphate mediated DNA transfer , Liposome mediated gene transfer , Potential target diseases for Gene Therapy , GENE THERAPY USED TO TREAT TYPE I DIABETES , GENE THERAPY FOR CANCER TREATMENT , PARKINSON’S DISEASE , GENE THERAPY TREATMENT FOR ADA-SCID , GENE THERAPY FOR CYSTIC FIBROSI S, HEMOPHILIA , BLINDNESS
Gene therapy , Nucleic acids , GENES , ADA-SCID(Severe combined immunodeficiency) , APPROACHES IN GENE THERAPY : Types of gene therapy , somatic gene therapy , germ line gene therapy , Gene modification , gene replacement , gene correction , gene augmentation , Gene transfer methods , viral gene transfer (biological) , non viral gene transfer : physical method ,chemical method , retrovirus vector system , adenovirus vector system , Adeno associated virus vector , Herpex simplex virus vector , Electroporation , Microinjection , Gene Gun , Calcium phosphate mediated DNA transfer , Liposome mediated gene transfer , Potential target diseases for Gene Therapy , GENE THERAPY USED TO TREAT TYPE I DIABETES , GENE THERAPY FOR CANCER TREATMENT , PARKINSON’S DISEASE , GENE THERAPY TREATMENT FOR ADA-SCID , GENE THERAPY FOR CYSTIC FIBROSI S, HEMOPHILIA , BLINDNESS
Vinay ju 5076 Virulence in plant pathogenic bacteria
Mutations and three mechanisms of genetic exchange allow bacteria to gain new genetic material: transformation, conjugation, and transduction. Conjugation involves the transfer of DNA between bacteria via a plasmid. Transduction involves the transfer of DNA between bacteria through bacteriophages. Studies have shown differences in virulence and presence of effector proteins among strains of Pseudomonas syringae isolated in California, and differences in plasmid profiles and copper resistance among strains of Pseudomonas syringae isolated from mango trees.
Gen therapy
Gene therapy seeks to modify or manipulate genes to treat diseases. There are three main strategies for gene therapy: gene augmentation therapy which introduces a normal gene to compensate for an abnormal gene, targeted killing of specific cells, and targeted inhibition of gene expression. Gene therapy can be somatic, affecting most body cells, or germline, affecting eggs or sperm. Viral vectors are commonly used to deliver genes into cells, but non-viral methods also exist. Gene editing tools like ZFNs, TALENs, and CRISPR-Cas systems can also be used to precisely alter genes.
Genetic polymorphism
Genetic and environmental factors are the two keys that make human phenotype variations. When the genomic DNA sequences on equivalent chromosomes of any two individuals are compared, there is substantial variation in the sequence at many points throughout the genome. The term polymorphism was originally used to describe variations in shape and form that distinguish normal individuals within a species from each other. These days, geneticists use the term genetic polymorphisms to describe the inter-individual, functionally silent differences in DNA sequence that make each human genome unique. In order to better understand the phenomenon of genetic polymorphism, an emphasis has been laid on the structures and functions of nucleotides, genes and nucleic acids, including their relationship with polymorphism.
Polymorphism can be caused by factors such as mutation, which is defined as a permanent transmissible change in DNA sequence. Mutations are classified based on where they occur somatic and germ line mutations) and the length of the nucleotide sequences they affect (gene-level and chromosomal mutations). The various types of polymorphisms include; single nucleotide polymorphisms (SNPs), small-scale insertions/deletions, polymorphic repetitive elements, microsatellite variation and haplotypes.
Variations in DNA sequences may have a major impact on how human beings respond to disease, bacteria, viruses, toxins, chemicals, drugs, and other therapies. Many clinical phenotypes observed in diseases seem to have considerable genetic components.
Determining genetic polymorphism can be based on morphological, biochemical, and molecular types of information. However, molecular markers have advantages over other kinds, where they show genetic differences on a more detailed level without interferences from environmental factors, and where they involve techniques that provide fast results detailing genetic diversity. Some of the techniques used in studying polymorphisms include; PCR based techniques and techniques involving DNA based markers.
Key words: Genetic polymorphism, effects in a population,
Gene Transfer
The document discusses gene transfer and the genetic code. It states that the genetic code is nearly universal, allowing gene transfer between species. It outlines the basic process of gene transfer using plasmids, host cells, restriction enzymes, and DNA ligase. Specifically, the plasmid is cut with enzymes, foreign DNA is inserted, then the recombinant plasmid is placed in a host cell where the desired gene product is produced, as demonstrated by harvesting insulin from E. coli.
Yeast 2 hybrid system ppt by meera qaiser
The yeast two-hybrid system is a technique used to study protein-protein interactions. It involves creating two fusion proteins, one with a DNA-binding domain and one with an activation domain. If the proteins of interest interact, they will reconstitute a functional transcription factor that activates a reporter gene. This allows researchers to identify novel interactions and characterize known interactions, helping to further understand biological processes at the molecular level.
genetic variations
Genetic variations can arise in bacteria through several mechanisms:
1. Point mutations or DNA rearrangements can occur spontaneously during DNA replication.
2. DNA can be transferred between bacteria through transformation, transduction, or conjugation. Transformation involves uptake of naked DNA. Transduction involves transfer via bacteriophages. Conjugation involves direct contact between bacteria.
3. These mechanisms allow for acquisition of new genes and increase genetic diversity, which can provide advantages like antibiotic resistance but also influence bacterial pathogenesis.
Yeast two hybrid
The yeast two-hybrid system is used to identify protein-protein interactions. It involves fusing two interacting proteins to a DNA-binding domain and transcriptional activation domain. If the proteins interact, it brings the domains together and activates a reporter gene. This allows identification of novel interactions and domains involved. Some advantages are it occurs in vivo, can find weak interactions, and doesn't require purified proteins. Disadvantages include false positives and some proteins may not fold correctly in yeast.
stem cell based gen therapy
Stem-cell based gene therapy aims to genetically modify cells to resist HIV infection through three main strategies: 1) targeting cellular genes essential for viral replication like CCR5, 2) directly targeting HIV gene expression, and 3) introducing genes that interfere with HIV replication. Combination therapy employing multiple genetic modifications may help prevent viral escape. Engineering immunity against HIV through therapeutic vaccines or modified T-cells and stem cells shows promise in controlling infection.
Gene therapy
Gene therapy involves inserting genes into cells to prevent, alleviate or cure diseases caused by genetic defects. There are two main types of gene therapy - somatic cell gene therapy, which targets non-reproductive cells, and germline cell gene therapy, which targets reproductive cells and can be inherited. There are two main approaches - ex vivo gene therapy, where cells are modified outside the body, and in vivo gene therapy, where genes are modified inside the body. Viral and non-viral vectors are used to deliver genes to cells. Gene therapy has shown promise in treating diseases like SCID, cystic fibrosis, and cancer. While theoretically a permanent cure, gene therapy still faces challenges and high costs that limit widespread use.
15 Therapy Of Genetics Diseases
This document discusses various approaches for therapy of genetic diseases, including conventional and gene therapy approaches. Conventional approaches include dietary therapy, protein/enzyme replacement, pharmacal therapy, and surgery. Gene therapy involves the deliberate introduction of genetic material into human cells for therapeutic purposes, and can be done through somatic or germline cell gene therapy, as well as ex vivo or in vivo approaches. Viral vectors are commonly used to deliver the therapeutic gene to target cells.
Gene transfer mechanisms in bacteria
This document discusses three mechanisms of gene transfer in bacteria: conjugation, transformation, and transduction. Conjugation involves the direct transfer of DNA via a connection tube from a donor to recipient cell. Transformation occurs when environmental DNA is taken up by a competent cell. Transduction is when a bacteriophage transfers DNA from one bacterial cell to another. All three mechanisms are unidirectional and involve recombination of the transferred DNA into the recipient genome.
Biochemistry Poster
Alpha-1 Antitrypsin (α-1 AT) deficiency is a common genetic disorder that affects 1 in 2,000 individuals in the USA. Additionally, over 20 million people have been identified as carriers for this genetic disorder. In severe cases, α-1 AT deficiency can cause substantial lung and liver damage, which if left untreated could result in death and there are no current available treatments. Alpha-1 protein is produced in the liver, travels in the bloodstream and utilized in the lungs to protect healthy lung tissue from harmful destruction by elastase. A common single amino acid substitution, located at E342K (ATZ) was identified in α-1 AT deficient humans. When this specific mutation occurs two phenotypes can result: 1) ATZ can polymerize in the liver causing cellular toxicity 2) inhibits alpha-1 antitrypsin from inhibiting elastase which can result in lung disease. Currently; little is known about the cellular mechanisms that clear the accumulated proteins in the liver. Therefore, an investigative study utilizing C. elegans model of ATZ was performed in order to help determine the cellular mechanisms that dispose of accumulated proteins. Specifically RNA interference was utilized to knockdown expression of specific genes. This investigation examined genes involved in the heat-shock pathway (HSP), unfolded protein response (UPR), and insulin signaling pathway (IS). Phenotypic analysis including: embryonic lethality, protein aggregation expression, and longevity, was completed after knockdown of genes to determine effect on ATZ accumulation. Currently with our preliminary data suggests that the heat-shack pathway may play a role in ATZ accumulation. Determining the mechanism of protein accumulation in the investigation of C. elegans may lead to possible drug targets and therefore the development of a treatment which may alleviate those diagnosed with this disorder.
ppt1.pptx
This document describes a study characterizing Wnt-activated cells in the mouse intestine. The researchers used two Wnt reporter mouse lines (TOPGAL and BatGal) to identify cells receiving a Wnt signal. They found Wnt-activated cells were primarily restricted to the crypt base and highest in the proximal small intestine. Interestingly, most Wnt-activated cells did not overlap with transient amplifying cells and expressed the stem cell marker Musashi-1 but not other markers. Gamma irradiation increased the number of Wnt-activated crypt cells, demonstrating the reporters can detect changes in Wnt signaling. The findings provide new insights into the regulation of the intestinal stem cell hierarchy.
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GENE THERAPY AND ITS APPLICATION
GENE THERAPY: TYPES, METHODS, FACTORS AND STANDARDS AND ITS APPLICATION IN HEALTHCARE FIELD
INVIVO THERAPY AND EXVIVO THERAPY
CHEMICAL AND PHYSICAL METHODS TO CARRY ON GENE THERAPY
DEFECTIVE GENE IDENTIFICATION IN GENE THERAPY AND TREATMENT OF GENETICALLY AFFECTED GENE BY GENE THERAPY
Transduction and transformation
Transduction is the process by which DNA is transferred between bacteria via a virus. It involves a bacteriophage infecting a bacterial cell and using the cell's machinery to produce new virus particles, which may incorporate small fragments of bacterial DNA into new viral genomes. This allows genetic material to be transferred from one bacterium to another without direct contact when the new viruses infect other bacteria. Transduction differs from transformation in that the transferred DNA is protected from DNase inside the viral particle. It is a molecular biology tool for introducing foreign DNA into host genomes.
MFF Poster-GRBaV-AC.pptx
1. Researchers at Cornell University studied the transcriptome of Grapevine Red Blotch-associated Virus (GRBaV) which causes Grapevine Red Blotch Disease. 2. They successfully extracted RNA from infected grapevine leaves and detected several discrete viral RNA transcripts between 100-3,200 nucleotides in size using northern blot hybridization. 3. Preliminary 5’-RACE sequencing results aim to identify the 5’-ends of viral mRNAs to better understand GRBaV transcription strategies.
Dna transfer
This document discusses various methods of transfection, which is defined as the introduction of foreign DNA into eukaryotic cells. It describes transfection methods such as calcium phosphate transfection, liposome-mediated transfection, retroviral transfection, and electroporation. It provides details on how each method works and compares their strengths and weaknesses. Common transfection methods like calcium phosphate and liposomes are simple but have low efficiency, while retroviral transfection can generate stable cell lines but has limitations on DNA size. Electroporation is fast and applicable to many cell types.
polymorphism
Genetic polymorphisms are normal variations that occur in more than 1% of the population. Single nucleotide polymorphisms are the most common type, involving a change in a single DNA base pair. Polymorphisms can occur in coding and regulatory regions, and may affect protein function or expression levels. The interleukin-1 gene cluster contains polymorphisms that have been associated with increased severity of periodontitis, where individuals with certain IL-1 genotypes experience more rapid bone and tissue loss around teeth. Understanding genetic predispositions can help with early detection and prevention of more serious disease.
Genetic polymorphism and It's Applications
Genetic polymorphism
Genetic polymorphism is the inheritance of a trait controlled by a single genetic locus with two alleles, in which the least common allele has a frequency of about 1% or greater. Genetic polymorphism is a difference in DNA sequence among individuals, groups, or populations.
Types of polymorphisms
Protein/enzyme polymorphisms
In the early days of human genetics, majority of polymorphisms were those associated with proteins and enzymes. To detect the polymorphism and a person’s genotype, one performed assays for the gene product, i.e., the protein or enzyme produced by the genetic blueprint.
DNA polymorphisms
The large class of polymorphisms are those that detect Slight variations at the level of DNA nucleotides.
Single nucleotide polymorphisms
A single nucleotide polymorphism or SNP is a sequence of DNA on which humans vary by one and only one nucleotide . Because humans differ by one nucleotide per every thousand or so nucleotides, there are millions of SNPs scattered throughout the human genome.
Tandem repeat polymorphisms
A tandem repeat polymorphism consists of a series of nucleotides that are repeated in tandem (i.e., one time after another). The polymorphism consists of the number of repeats.
Restriction Fragment Length Polymorphism (RFLP)
Restriction Fragment Length Polymorphism (RFLP) is a type in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA. If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme.
Applications of Genetic Polymorphism
The study of polymorphism has many uses in medicine, biological research, and law enforcement. Genetic diseases may be caused by a specific polymorphism. Scientists can look for these polymorphisms to determine if a person will develop the disease, or risks passing it on to his or her children.
Gene Therapy, Applications and Recent advances
This document discusses gene therapy and is presented by Urvashi Shakarwal. It defines gene therapy as the insertion of genes into an individual's cells to treat a disease. It describes the different approaches to gene therapy, including viral and non-viral vectors. Viral vectors commonly used are retroviruses, adenoviruses, and herpes simplex viruses. Non-viral methods include electroporation, microinjection, gene guns, and calcium phosphate or liposome transfection. The document outlines several applications of gene therapy, such as for chronic granulomatosis disorder, hemophilia, and inherited blindness.
Genetic polymorphism
The document discusses genetic polymorphisms in Plasmodium falciparum, the parasite that causes malaria. It defines key terms like locus, allele, and genome. It then describes different types of genetic polymorphisms like single nucleotide polymorphisms (SNPs), insertions and deletions (INDELs), and short tandem repeats (STRs). The document focuses on polymorphisms related to drug resistance in P. falciparum, discussing genes associated with resistance to chloroquine (pfcrt) and other antimalarial drugs, along with specific mutations in those genes linked to resistance.
Gene therapy
Gene therapy , Nucleic acids , GENES , ADA-SCID(Severe combined immunodeficiency) , APPROACHES IN GENE THERAPY : Types of gene therapy , somatic gene therapy , germ line gene therapy , Gene modification , gene replacement , gene correction , gene augmentation , Gene transfer methods , viral gene transfer (biological) , non viral gene transfer : physical method ,chemical method , retrovirus vector system , adenovirus vector system , Adeno associated virus vector , Herpex simplex virus vector , Electroporation , Microinjection , Gene Gun , Calcium phosphate mediated DNA transfer , Liposome mediated gene transfer , Potential target diseases for Gene Therapy , GENE THERAPY USED TO TREAT TYPE I DIABETES , GENE THERAPY FOR CANCER TREATMENT , PARKINSON’S DISEASE , GENE THERAPY TREATMENT FOR ADA-SCID , GENE THERAPY FOR CYSTIC FIBROSI S, HEMOPHILIA , BLINDNESS
Gene therapy , Nucleic acids , GENES , ADA-SCID(Severe combined immunodeficiency) , APPROACHES IN GENE THERAPY : Types of gene therapy , somatic gene therapy , germ line gene therapy , Gene modification , gene replacement , gene correction , gene augmentation , Gene transfer methods , viral gene transfer (biological) , non viral gene transfer : physical method ,chemical method , retrovirus vector system , adenovirus vector system , Adeno associated virus vector , Herpex simplex virus vector , Electroporation , Microinjection , Gene Gun , Calcium phosphate mediated DNA transfer , Liposome mediated gene transfer , Potential target diseases for Gene Therapy , GENE THERAPY USED TO TREAT TYPE I DIABETES , GENE THERAPY FOR CANCER TREATMENT , PARKINSON’S DISEASE , GENE THERAPY TREATMENT FOR ADA-SCID , GENE THERAPY FOR CYSTIC FIBROSI S, HEMOPHILIA , BLINDNESS
Vinay ju 5076 Virulence in plant pathogenic bacteria
Mutations and three mechanisms of genetic exchange allow bacteria to gain new genetic material: transformation, conjugation, and transduction. Conjugation involves the transfer of DNA between bacteria via a plasmid. Transduction involves the transfer of DNA between bacteria through bacteriophages. Studies have shown differences in virulence and presence of effector proteins among strains of Pseudomonas syringae isolated in California, and differences in plasmid profiles and copper resistance among strains of Pseudomonas syringae isolated from mango trees.
Gen therapy
Gene therapy seeks to modify or manipulate genes to treat diseases. There are three main strategies for gene therapy: gene augmentation therapy which introduces a normal gene to compensate for an abnormal gene, targeted killing of specific cells, and targeted inhibition of gene expression. Gene therapy can be somatic, affecting most body cells, or germline, affecting eggs or sperm. Viral vectors are commonly used to deliver genes into cells, but non-viral methods also exist. Gene editing tools like ZFNs, TALENs, and CRISPR-Cas systems can also be used to precisely alter genes.
Genetic polymorphism
Genetic and environmental factors are the two keys that make human phenotype variations. When the genomic DNA sequences on equivalent chromosomes of any two individuals are compared, there is substantial variation in the sequence at many points throughout the genome. The term polymorphism was originally used to describe variations in shape and form that distinguish normal individuals within a species from each other. These days, geneticists use the term genetic polymorphisms to describe the inter-individual, functionally silent differences in DNA sequence that make each human genome unique. In order to better understand the phenomenon of genetic polymorphism, an emphasis has been laid on the structures and functions of nucleotides, genes and nucleic acids, including their relationship with polymorphism.
Polymorphism can be caused by factors such as mutation, which is defined as a permanent transmissible change in DNA sequence. Mutations are classified based on where they occur somatic and germ line mutations) and the length of the nucleotide sequences they affect (gene-level and chromosomal mutations). The various types of polymorphisms include; single nucleotide polymorphisms (SNPs), small-scale insertions/deletions, polymorphic repetitive elements, microsatellite variation and haplotypes.
Variations in DNA sequences may have a major impact on how human beings respond to disease, bacteria, viruses, toxins, chemicals, drugs, and other therapies. Many clinical phenotypes observed in diseases seem to have considerable genetic components.
Determining genetic polymorphism can be based on morphological, biochemical, and molecular types of information. However, molecular markers have advantages over other kinds, where they show genetic differences on a more detailed level without interferences from environmental factors, and where they involve techniques that provide fast results detailing genetic diversity. Some of the techniques used in studying polymorphisms include; PCR based techniques and techniques involving DNA based markers.
Key words: Genetic polymorphism, effects in a population,
Gene Transfer
The document discusses gene transfer and the genetic code. It states that the genetic code is nearly universal, allowing gene transfer between species. It outlines the basic process of gene transfer using plasmids, host cells, restriction enzymes, and DNA ligase. Specifically, the plasmid is cut with enzymes, foreign DNA is inserted, then the recombinant plasmid is placed in a host cell where the desired gene product is produced, as demonstrated by harvesting insulin from E. coli.
Yeast 2 hybrid system ppt by meera qaiser
The yeast two-hybrid system is a technique used to study protein-protein interactions. It involves creating two fusion proteins, one with a DNA-binding domain and one with an activation domain. If the proteins of interest interact, they will reconstitute a functional transcription factor that activates a reporter gene. This allows researchers to identify novel interactions and characterize known interactions, helping to further understand biological processes at the molecular level.
genetic variations
Genetic variations can arise in bacteria through several mechanisms:
1. Point mutations or DNA rearrangements can occur spontaneously during DNA replication.
2. DNA can be transferred between bacteria through transformation, transduction, or conjugation. Transformation involves uptake of naked DNA. Transduction involves transfer via bacteriophages. Conjugation involves direct contact between bacteria.
3. These mechanisms allow for acquisition of new genes and increase genetic diversity, which can provide advantages like antibiotic resistance but also influence bacterial pathogenesis.
Yeast two hybrid
The yeast two-hybrid system is used to identify protein-protein interactions. It involves fusing two interacting proteins to a DNA-binding domain and transcriptional activation domain. If the proteins interact, it brings the domains together and activates a reporter gene. This allows identification of novel interactions and domains involved. Some advantages are it occurs in vivo, can find weak interactions, and doesn't require purified proteins. Disadvantages include false positives and some proteins may not fold correctly in yeast.
stem cell based gen therapy
Stem-cell based gene therapy aims to genetically modify cells to resist HIV infection through three main strategies: 1) targeting cellular genes essential for viral replication like CCR5, 2) directly targeting HIV gene expression, and 3) introducing genes that interfere with HIV replication. Combination therapy employing multiple genetic modifications may help prevent viral escape. Engineering immunity against HIV through therapeutic vaccines or modified T-cells and stem cells shows promise in controlling infection.
Gene therapy
Gene therapy involves inserting genes into cells to prevent, alleviate or cure diseases caused by genetic defects. There are two main types of gene therapy - somatic cell gene therapy, which targets non-reproductive cells, and germline cell gene therapy, which targets reproductive cells and can be inherited. There are two main approaches - ex vivo gene therapy, where cells are modified outside the body, and in vivo gene therapy, where genes are modified inside the body. Viral and non-viral vectors are used to deliver genes to cells. Gene therapy has shown promise in treating diseases like SCID, cystic fibrosis, and cancer. While theoretically a permanent cure, gene therapy still faces challenges and high costs that limit widespread use.
15 Therapy Of Genetics Diseases
This document discusses various approaches for therapy of genetic diseases, including conventional and gene therapy approaches. Conventional approaches include dietary therapy, protein/enzyme replacement, pharmacal therapy, and surgery. Gene therapy involves the deliberate introduction of genetic material into human cells for therapeutic purposes, and can be done through somatic or germline cell gene therapy, as well as ex vivo or in vivo approaches. Viral vectors are commonly used to deliver the therapeutic gene to target cells.
Gene transfer mechanisms in bacteria
This document discusses three mechanisms of gene transfer in bacteria: conjugation, transformation, and transduction. Conjugation involves the direct transfer of DNA via a connection tube from a donor to recipient cell. Transformation occurs when environmental DNA is taken up by a competent cell. Transduction is when a bacteriophage transfers DNA from one bacterial cell to another. All three mechanisms are unidirectional and involve recombination of the transferred DNA into the recipient genome.
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Vinay ju 5076 Virulence in plant pathogenic bacteria
Vinay ju 5076 Virulence in plant pathogenic bacteria
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Biochemistry Poster
Alpha-1 Antitrypsin (α-1 AT) deficiency is a common genetic disorder that affects 1 in 2,000 individuals in the USA. Additionally, over 20 million people have been identified as carriers for this genetic disorder. In severe cases, α-1 AT deficiency can cause substantial lung and liver damage, which if left untreated could result in death and there are no current available treatments. Alpha-1 protein is produced in the liver, travels in the bloodstream and utilized in the lungs to protect healthy lung tissue from harmful destruction by elastase. A common single amino acid substitution, located at E342K (ATZ) was identified in α-1 AT deficient humans. When this specific mutation occurs two phenotypes can result: 1) ATZ can polymerize in the liver causing cellular toxicity 2) inhibits alpha-1 antitrypsin from inhibiting elastase which can result in lung disease. Currently; little is known about the cellular mechanisms that clear the accumulated proteins in the liver. Therefore, an investigative study utilizing C. elegans model of ATZ was performed in order to help determine the cellular mechanisms that dispose of accumulated proteins. Specifically RNA interference was utilized to knockdown expression of specific genes. This investigation examined genes involved in the heat-shock pathway (HSP), unfolded protein response (UPR), and insulin signaling pathway (IS). Phenotypic analysis including: embryonic lethality, protein aggregation expression, and longevity, was completed after knockdown of genes to determine effect on ATZ accumulation. Currently with our preliminary data suggests that the heat-shack pathway may play a role in ATZ accumulation. Determining the mechanism of protein accumulation in the investigation of C. elegans may lead to possible drug targets and therefore the development of a treatment which may alleviate those diagnosed with this disorder.
ppt1.pptx
This document describes a study characterizing Wnt-activated cells in the mouse intestine. The researchers used two Wnt reporter mouse lines (TOPGAL and BatGal) to identify cells receiving a Wnt signal. They found Wnt-activated cells were primarily restricted to the crypt base and highest in the proximal small intestine. Interestingly, most Wnt-activated cells did not overlap with transient amplifying cells and expressed the stem cell marker Musashi-1 but not other markers. Gamma irradiation increased the number of Wnt-activated crypt cells, demonstrating the reporters can detect changes in Wnt signaling. The findings provide new insights into the regulation of the intestinal stem cell hierarchy.
1041
Bright is a transcription factor required for both hematopoietic stem cell development and B cell lineage development. Mice lacking Bright die by mid-gestation due to failed hematopoiesis. Rare Bright-deficient mice that survive have deficits in hematopoietic stem cells and impaired B cell development and function, including reduced natural antibody and IgG1 class switching responses. Bright is thus necessary for both the formation of hematopoietic stem cells and their differentiation into specific blood lineages such as B cells.
12. Cell synchronization and Immortalization
Cell synchronization helps in obtaining distinct sub population of cells representing different stages of cell cycle.It helps in collecting population wide data of cells progressing through various stages of cell cycle. Immortalization, refers to cells having capability of undergoing cell division infinitely. Immortal cells are particularly preferred in cell culture to enable long time storage and use. This presentation teaches about cell synchronization, methods of cell synchronization, cellular transformation, immortalization and mechanism of immortalization.
Cell line development
This document discusses various methods for selecting and developing cell lines for research. It covers primary culture isolation, subculture propagation to establish a cell line, control of cell proliferation through growth factors and intracellular regulators, senescence limits on cell divisions, differentiation inhibiting proliferation, and the need for continuous cell lines. Methods to immortalize cell lines include viral genes like SV40 LT and HPV E6/E7 to inactivate tumor suppressors, adenoviral and retroviral vectors, telomerase induction with hTERT, use of oncogenes, and cell hybridization. The goal is to generate stable, consistent cell lines that proliferate indefinitely while maintaining similar phenotype to the original tissue.
Samuel Dugger FGF8b Final Report
This document describes research aimed at isolating an aptamer that can detect the fibroblast growth factor FGF8b, which has been linked to prostate cancer. The researcher conducted four rounds of SELEX (Systematic Evolution of Ligands by EXponential enrichment) using bead-based selection to isolate RNA aptamers that bind specifically to FGF8b. No aptamers had previously been found for this target. Successfully finding an FGF8b-binding aptamer could help detect prostate cancer earlier and improve treatment outcomes.
Inhibitors of Microtubule Polymerization
This document describes a screen of 50,000 compounds to identify inhibitors of hepatocyte growth factor (HGF)-induced epithelial scattering. Several structurally distinct compounds were identified that had not previously been reported to have biological activity. Further analysis revealed that many of the compounds broadly inhibit cancer cell proliferation by arresting cell division in G2/M phase with minimal induction of apoptosis. This effect is consistent with microtubule-targeting agents. Biochemical assays showed that several compounds directly inhibit microtubule polymerization. Some pharmacokinetic properties of the compounds were also assessed.
Dissertation final complete1
This document summarizes a study investigating the epigenetic changes that occur during the transdifferentiation of pancreatic acinar cells (HPACs) to hepatocyte-like cells. The study found that DNA methylation in HPACs peaks at 24 hours after treatment with dexamethasone, and the cells display phenotypic changes associated with hepatocytes after 7 days. The study also examined the promoter sequence of the SGK1 gene, which is involved in the transdifferentiation process. The results suggest therapeutic potential for producing hepatocytes from pancreatic cells to treat liver disease in a more cost-effective manner than current alternatives.
Prevention of lysosomal storage diseases and derivation of2
This study describes the use of preimplantation genetic diagnosis (PGD) to prevent the transmission of four lysosomal storage disorders: Tay-Sachs disease, Gaucher disease, Fabry disease, and Hunter syndrome. PGD was performed on 28 embryos from 9 couples, identifying 2 embryos with mutations and deriving stem cell lines from them. The technique was highly accurate, avoiding transmission of diseases while establishing mutant stem cell lines for research.
Xenotransplantation of pig kidney to human
The document discusses strategies for xenotransplantation using genetically modified pigs. It notes that over 18 patients die each day waiting for organ transplants. Pigs are a promising donor species due to anatomical similarities to humans. However, immunological barriers like hyperacute and acute cellular rejection must be overcome. Methods discussed include genetically knocking out GGTA1 and CMAH genes to eliminate antigen epitopes and introducing human complement regulatory genes. Genome editing tools like ZFNs, CRISPR-Cas9, and SCNT are used to generate multitransgenic pigs lacking immunogenic antigens and capable of preventing rejection. Studies produced GGTA1 knockout piglets and fetuses lacking the alpha-Gal epitope using these techniques.
Prevention of lysosomal storage diseases and derivation of2
This study evaluated the use of preimplantation genetic diagnosis (PGD) to prevent the transmission of four lysosomal storage disorders (TSD, GD, FD, HS) in couples at risk. PGD was performed on 28 embryos from 8 couples, identifying 2 embryos with mutations for Hunter syndrome and GD. These embryos were used to derive 2 human embryonic stem cell lines expressing markers of an undifferentiated state. PGD accurately diagnosed mutations in embryos and allowed derivation of disease-specific stem cell lines, demonstrating the potential of PGD to avoid inherited diseases while generating disease models.
Higher expression of SATB2 in hepatocellular carcinoma of African Americans d...
Abstract
In the United States, Hepatocellular Carcinoma (HCC) incidence has tripled over the past two decades. The disease has disproportionately affected minority and disadvantaged
populations. The purpose of this study was to examine the expression of SATB2 gene in HCC cells derived from African Americans (AA) and Caucasian Americans (CA) and assess its oncogenic potential by measuring cell viability, spheroid
formation, epithelial‐mesenchymal transition (EMT), stem cell markers and pluripotency maintaining factors in cancer stem cells (CSCs). We compared the expression of SATB2 in human primary hepatocytes, HCC cells derived from AA and CA, and
HCC CSCs. Hepatocellular carcinoma cells derived from AA expressed the higher level of SATB2 than those from CA. By comparison, normal human hepatocytes did
not express SATB2. Higher expression of SATB2 in HCC cells from AA was associated with greater growth rate, cell viability, colony formation and EMT characteristics than those from CA. Knockout of SATB2 in CSCs by Crispr/Cas9 technique significantly inhibited the expression of SATB2 gene, stem cell markers (CD24, CD44 and CD133), pluripotency maintaining factors (c‐Myc, KLF4, SOX2 and OCT4), and EMT
compared with non‐targeting control group. The expression of SATB2 was negatively correlated with miR34a. SATB2 rescued the miR‐34a‐mediated inhibition of CSC's viability. These data suggest that SATB2 is an oncogenic factor, and its higher expression may explain the disparity in HCC outcomes among AA.
Wei Yu1 | Sanjit K. Roy2 | Yiming Ma1 | Thomas A. LaVeist3 | Sharmila Shankar1,2,4 |
Rakesh K. Srivastava1,2,4
ppt1.pptx
This document summarizes a study characterizing Wnt signaling activity in the mouse intestine during homeostasis. The researchers used two mouse lines expressing Wnt reporters to identify intestinal epithelial cells actively receiving Wnt signals. They found that Wnt-activated cells were primarily located at the base of crypts and expressed the stem cell marker Musashi-1, but not markers of transient amplifying cells or differentiated cells. They also showed that gamma irradiation increased the number of Wnt-activated crypt cells, demonstrating the reporters can detect changes in Wnt signaling after injury. The study provides new insights into the regulation of intestinal stem cell hierarchy during homeostasis and disease.
Summer Conference 2016
This study examined the effects of inhibiting the Hippo signaling pathway during sea urchin embryogenesis. Researchers treated sea urchin embryos with verteporfin, an inhibitor of YAP which is a key component of the Hippo pathway. They found that inhibiting the Hippo pathway delayed cell cleavage and gastrulation. While marker genes for specific cell types were still expressed, the patterns of expression were altered. Inhibition of the Hippo pathway resulted in epithelial cells that were larger in size and fewer in number, suggesting YAP is involved in epithelial organization and proliferation during sea urchin development.
072412 high school teachers 2
The document introduces the budding yeast Saccharomyces cerevisiae as a model organism for genetics studies, describing its life cycle between haploid and diploid forms and advantages like its small genome and rapid growth. Methods for conducting genetic screens in yeast are discussed, including how early work isolating cell cycle genes used temperature-sensitive mutants and screening for cell cycle arrest. Meiosis and sporulation in yeast allows studying meiotic divisions and genetic analysis by ensuring mutations are present in only one gene.
072412 high school teachers 2
The document introduces the budding yeast Saccharomyces cerevisiae as a model organism for genetics studies, describing its life cycle between haploid and diploid forms and advantages like its small genome and rapid growth. Methods for conducting genetic screens in yeast are discussed, including how early work isolating cell cycle genes used temperature-sensitive mutants and screening for cell cycle arrest. Meiosis and sporulation in yeast allows studying meiotic divisions and genetic analysis by ensuring mutations are present in only one gene.
Nuhu et al_Poster NAPA2016 correction and observation
The study determined the prevalence and genetic profiles of ESBL-producing uropathogens among members of the Enterobacteriaceae family at Specialist Hospital Sokoto, Nigeria. A total of 64 Gram-negative uropathogens were isolated from 365 urine samples, with E. coli and Salmonella arizonae being most prevalent. The isolates showed high resistance to cotrimoxazole, nalidixic acid, ciprofloxacin and norfloxacin. 64.1% of isolates were multidrug resistant. ESBL production was detected in 23.4% of isolates. PCR analysis showed 73.3% of ESBL producers contained the blaCTX-M gene and 26.7
Evolution of Pathogens
The document discusses genome evolution in bacterial pathogens. It describes two main ways that phenotypic similarities can evolve between species: parallel evolution where similar mutations arise independently, and collateral evolution where alleles are shared between populations. It provides examples of how pathogenic strains like Shigella arose from E. coli through plasmid acquisition and phenotypic convergence. The document also summarizes key points from a workshop on bacterial pathogen origin and evolution, including the four main forms of genome evolution and the role of horizontal gene transfer in transmitting virulence genes.
Blood Group Genotyping
Genomics is impacting all areas of medicine. In transfusion medicine, DNA-based genotyping is being used as an alternative to serological antibody-based methods.
Single nucleotide changes (SNP's) in the respective genes are responsible for most antigenic polymorphisms.
Validated by comparison with antibody-based typing.
Provide the patient’s comprehensive extended blood group profile as part of their medical record by the growth of whole-genome sequencing (WGS).
Prevention of Lysosomal Storage Diseases and Derivation of Mutant StemCell Li...
Preimplantation genetic diagnosis was used to avoid the transmission of four lysosomal storage disorders (TSD, GD, FD, HS) in couples at risk. 329 eggs from 56 cycles underwent PGD and analysis, resulting in 20 families avoiding transmission. Two human embryonic stem cell lines were derived from embryos diagnosed with Hunter syndrome and Gaucher disease. The cell lines expressed stem cell markers and had a normal karyotype. PGD is an effective technique for avoiding genetic transmission of lysosomal storage disorders and generating disease-specific stem cell lines.
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Prevention of lysosomal storage diseases and derivation of2
Prevention of lysosomal storage diseases and derivation of2
Prevention of lysosomal storage diseases and derivation of2
Prevention of lysosomal storage diseases and derivation of2
Higher expression of SATB2 in hepatocellular carcinoma of African Americans d...
Higher expression of SATB2 in hepatocellular carcinoma of African Americans d...
Nuhu et al_Poster NAPA2016 correction and observation
Nuhu et al_Poster NAPA2016 correction and observation
Prevention of Lysosomal Storage Diseases and Derivation of Mutant StemCell Li...
Prevention of Lysosomal Storage Diseases and Derivation of Mutant StemCell Li...
STEP_UP poster test 8
- 1. w Screening for Regulators of Quiescence in the Drosophila Testis Stem Cell Niche Linh Pham1 , Leah Greenspan2 ,Margaret de Cuevas2 , Erika Matunis2 1 Humboldt State University, Department of Biology; 2 Johns Hopkins University, Department of Cell Biology 5. Results 9. References 7. Conclusions and Impacts • Fly progeny at 18°C • RNAi inactive • Phenotype not expressed • Adult flies shifted to 31°C; 7days • RNAi active • Phenotype expressed Testes dissection Fluorescence immunostaining of testes Imaging testes with confocal microscope Whole mounting testes 10. Acknowledgements Pathway 6 Number of components knocked down Location of knock down Number of testes total Hub quiescence changed? JAK/STAT 3 Hub; Stem Cells Hub: 49 ; Stem cells: 14 No EGFR 2 Hub; Stem Cells Hub: 24 ; Stem cells: 48 No Wg 1 Hub 27 Yes Hub: Quiescent 1. Introduction 3. Methods • Stem cells can differentiate into specialized cells or self-renew. • Microenvironment where stem and niche cells exist: niche. • Niche cells tell stem cells when to differentiate or self-renew. • Drosophila (fruit fly) testis provides a simple model to observe niche interactions. • Niche cells are known as hub cells in Drosophila testis. • In wild-type flies, hub cells are non-dividing (quiescent). • When stem cells are eliminated (through damage), hub cells become mitotic and change into stem cells 3 . • When niche signals are misregulated, this can lead to various diseases (i.e. cancer) 5 . • Understanding basics of niche regulation is important for combating these diseases. Wild-type hub RBF-1 reduced in hub • Retinoblastoma Family Protein-1 (RBF-1), a cell cycle inhibitor, also produced mitotic, enlarged hubs, when RBF-1 was knocked down in hub 2 . • This indicates that niche signaling may be responsible for regulating hub quiescence. 2. Hypothesis Extracellular signals in the niche interact with signaling pathway receptors on hub cells to, like RBF, maintain hub quiescence. • Candidate pathways for maintenance of hub quiescence were perturbed • Changes in hub and testis were observed. • Perturbation involved reducing (RNAi knockdown) receptors or ligands of candidate pathways. • A temperature shift method (Gal4-UAS/Gal80 TS ) induced knockdown. 4. Experimental Layout 1.The majority of pathways tested did not change hub quiescence. • JAK/STAT and EGFR are conserved stem cell maintenance pathways 4 . • Wg is the fly homolog of ligand Wnt from the Wnt pathway. This pathway is known to cause colorectal cancer when misregulated in humans 1 . Wg RNAi in hub Hub and Mitotic Cells Germ Cells • Fig. A: One mitotic cell is seen in the hub (dense green nucleus) • Fig. B: Green channel showing only hub and mitotic cell • Fig. C: Red channel showing only germ cells (including stem cells). • Mitotic cell’s location is overlaid with pink line in Fig. B and Fig. C. • Note that this location is not marked red in Fig. C, indicating that mitotic cell is probably a hub cell and not a stem/germ cell. • Extracellular signals in the niche may, in fact, interact with hub receptors to maintain hub quiescence. • Signals regulating hub quiescence are most likely very specific, for only one pathway tested produced a mitotic hub cell. • Identifying signaling pathways that regulate hub quiescence could provide the basis for understanding diseases relating to misregulation of niche signaling. 1. Knocking down Wg in hub for 2 weeks may provide an interesting hub phenotype. 8. Future Directions 2. Knocking down more components of the Wg pathway and observing hub phenotype. Weak Phenotype 2 weeks • Longer experimenting time may allow more hub cells to divide, leading to enlarged hubs. • Because mitosis is only a short stage in the cell cycle, probability of staining for a mitotic hub increases if an experiment is repeated for more replicates. 3. Knocking down pathway components in combinations to confirm negative results. 1 week • Knocking down the ligand Wg produced a mitotic hub. • If knocking down receptor and downstream components give the same phenotype, this would confirm that Wg is truly involved in regulating hub quiescence • Knocking down only the ligand or receptor of EGFR and JAK/STAT may not disrupt strongly enough to induce mitosis in hub. • Knocking down ligand and receptor together could result in stronger disruptions of the pathways. • If hub quiescence is regulated by either of these pathways, this stronger disruption will most likely produce a hub phenotype. DAPI (nuclei); Fas3 (hub membrane); PH3 (mitotic cells); Vasa (germ cells) ligand receptor A B C DAPI (nuclei); Fas3 (hub membrane); PH3 (mitotic cells); Vasa (germ cells) 2. Knocking down Wg in hub produced 1 hub (of 27) with a mitotic cell 1.Bienz M, Clevers H.(2000). Linking colorectal cancer to Wnt signaling. Cell 103(2), 311-20. 2.Giacinti C, Giordano A (2006). RB and cell cycle progression.Oncogene. 25, 5220-7 3.Hetie, P., et al. (2014). Conversion of Quiescent Niche Cells to Somatic Stem Cells Causes Ectopic Niche Formation in the Drosophila Testis. Cell Reports 7, 715-21. 4.Lucchetta EM, Ohstein B. (2012). The Drosophila midgut: a model for stem cell driven tissue regeneration.Wiley Interdiscip Rev Dev Biol.1(5), 781-8. 5.Reya T, et al. (2001). Stem cells, cancer, and cancer stem cells. Nature 414, 105–11. 6. RNAi lines: Stat92e (43866,106980), upd (3282), dome (106071), EGFR (107130 ), spitz (34645), Wg (13352); Hub driver: E132; CySC driver: C587. Strong Phenotype? I would like to thank STEP-UP for funding this opportunity, and members of Dr. Matunis's lab for their patience and support throughout this project. Zel Demere Tiffaney Tran Normal Damaged