Gene Expression Methods and E. coli Systems
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Questions and Answers

What is the purpose of blotting techniques in molecular genetics?

  • To measure gene expression levels quantitatively
  • To detect specific DNA fragments in complex mixtures (correct)
  • To analyze protein structures
  • To amplify DNA sequences

    • What do microarray techniques primarily reveal?

  • mRNA expression, co-regulation, and localization (correct)
  • Protein-protein interactions
  • DNA replication processes
  • Cellular metabolic pathways

    • What is a characteristic of recombinant DNA expression vectors?

  • They facilitate uncontrolled exogenous gene expression
  • They solely exist in eukaryotic cells
  • They enable regulated expression of exogenous genes (correct)
  • They are only used for gene editing

    • What is the primary function of in situ hybridization in developmental biology?

    <p>To detect the activity of specific genes in whole and sectioned embryos</p> Signup and view all the answers

    Which of the following techniques is NOT used for gene expression analysis?

    <p>DNA sequencing</p> Signup and view all the answers

    What does a green spot indicate on a DNA microarray?

    <p>Higher expression in serum-starved cells.</p> Signup and view all the answers

    What is represented by a red spot on a DNA microarray?

    <p>Higher expression in serum(+) cells.</p> Signup and view all the answers

    What does a yellow spot on a DNA microarray indicate?

    <p>Equal hybridization of green and red fluorescence.</p> Signup and view all the answers

    What is one benefit of using E. coli expression systems?

    <p>They can produce large quantities of proteins from cloned genes.</p> Signup and view all the answers

    Which component is essential in the E. coli expression vector for gene transcription?

    <p>The lac promoter.</p> Signup and view all the answers

    Which characteristic is NOT true about the expression vector plasmid used in E. coli?

    <p>Produces proteins at low quantities.</p> Signup and view all the answers

    What type of cells can produce eukaryotic proteins from plasmid vectors?

    <p>Both bacterial and eukaryotic cells.</p> Signup and view all the answers

    What does the presence of the lacZ gene in the expression vector indicate?

    <p>It enables the detection of successful transformation.</p> Signup and view all the answers

    What is the primary function of the lacZ gene in E. coli?

    <p>To produce β-galactosidase</p> Signup and view all the answers

    What is the significance of using nitrophenyl-β-D-galactopyraniside (ONPG) in assays?

    <p>It is a substrate that releases a colored product upon cleavage</p> Signup and view all the answers

    How does β-galactosidase activity measured in transfected cells?

    <p>By tracking the increase in absorbance at 420 nm</p> Signup and view all the answers

    Which of the following statements about β-galactosidase is true?

    <p>It produces stable protein products</p> Signup and view all the answers

    What is measured to assess β-galactosidase activity during an assay?

    <p>Absorbance at 420 nm</p> Signup and view all the answers

    In the context of the lac operon, which condition is necessary for the action of β-galactosidase?

    <p>Presence of lactose or a lactose analog</p> Signup and view all the answers

    What is a key characteristic of lentivirus delivery in gene therapy?

    <p>It integrates DNA into the genome.</p> Signup and view all the answers

    Which of the following best describes the lysogenic effect of β-galactosidase in genetic experiments?

    <p>It allows for easy detection of gene expression</p> Signup and view all the answers

    What color is produced when β-galactosidase cleaves ONPG?

    <p>Yellow</p> Signup and view all the answers

    Which viral coat protein is mentioned as part of the hybrid virus particles?

    <p>VSV-G</p> Signup and view all the answers

    What is the carrying capacity of lentivirus mentioned in the content?

    <p>8-kb</p> Signup and view all the answers

    What type of cells can lentivirus infect?

    <p>A wide variety of mammalian cells</p> Signup and view all the answers

    How does recombinant lentivirus particle production occur?

    <p>By transient transfection of three different plasmids</p> Signup and view all the answers

    What is the role of lentivirus retroviruses in gene modification?

    <p>They increase the efficiency of gene integration and expression.</p> Signup and view all the answers

    What type of cells can the lentivirus infect according to the options provided?

    <p>Any mammalian target cell types</p> Signup and view all the answers

    Why is lentivirus widely used in neuroscience experiments?

    <p>It allows for long-term gene expression.</p> Signup and view all the answers

    What is the primary function of the first plasmid in recombinant lentivirus particle production?

    <p>Controlling the expression of the cloned gene of interest</p> Signup and view all the answers

    Which of the following is NOT a role of the LTR sequences in the system described?

    <p>Acting as a selectable marker for transformed cells</p> Signup and view all the answers

    What does the packaging plasmid accomplish in the recombinant lentivirus particle production process?

    <p>It carries the viral genes required for assembling the viral particle.</p> Signup and view all the answers

    What is the role of the second plasmid in the lentivirus production system?

    <p>To provide the necessary components for viral particle packaging</p> Signup and view all the answers

    Which mammalian target cell types can the lentivirus particles infect due to the viral coat protein used?

    <p>A variety of cell types including stem cells, neurons, and liver cells</p> Signup and view all the answers

    What initiates the conversion of RNA to double-stranded DNA in the target cell?

    <p>LTR sequences</p> Signup and view all the answers

    Which component of the recombinant lentivirus system is responsible for enabling a broader range of infections across various cell types?

    <p>The viral coat plasmid</p> Signup and view all the answers

    Why is the neor selectable marker significant in the context of the first plasmid?

    <p>It allows for the identification of successfully transfected cells.</p> Signup and view all the answers

    Study Notes

    Gene Expression Methods

    • DNA microarray analysis is used to compare gene expression in different conditions.
    • Green spots indicate higher expression in serum-starved cells.
    • Red spots indicate higher expression in serum-treated cells.
    • Yellow spots indicate no change in gene expression.
    • In situ hybridization can detect the activity of specific genes in embryos.
    • Recombinant DNA expression vectors allow regulated gene expression and protein production in both bacteria and eukaryotic cells.

    E. coli Expression Systems

    • E. coli expression systems use plasmids with the lac promoter and lacZ gene to produce large quantities of protein.
    • The lacZ gene codes for β-galactosidase, an enzyme that cleaves ONPG (nitrophenyl-β-D-galactopyraniside) in a buffer containing sodium phosphate and magnesium chloride.
    • The cleavage of ONPG by β-galactosidase releases o-nitrophenol, which is yellow and absorbs 420 nm light.
    • Monitoring the accumulation of yellow color (increased 420 nm absorbance) provides a measurement of β-galactosidase activity.

    Lentivirus Delivery

    • Lentiviruses are retroviruses that can integrate modified genes into animal cell genomes for stable expression.
    • Lentivirus delivery is efficient for a variety of cell types, including hematopoietic stem cells, neurons, muscle, and liver cells.

    Lentivirus Particle Production

    • Three plasmids are used for recombinant lentivirus particle production:
      • Vector plasmid: contains the cloned gene of interest, a selectable marker (e.g., neor), and lentivirus LTR sequences.
      • Packaging plasmid: carries the viral genes (except the major coat protein) needed for packaging LTR-containing RNA.
      • Viral coat plasmid: encodes a viral coat protein (e.g., VSV-G) that allows the virus to infect a wide range of cell types.

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    Molecular Genetic Techniques PDF

    Description

    Explore the various methods used in gene expression analysis, focusing on DNA microarray analysis and in situ hybridization. Learn about E. coli expression systems, including the use of plasmids and the lac operon for protein production. This quiz will test your understanding of key concepts and techniques in molecular biology.

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