GQ

DNA Cloning and Gene Expression

DNA Cloning and Gene Expression

  • Central dogma of molecular biology: DNA → RNA → Protein
    • Transcription: DNA is transcribed into RNA by RNA polymerase.
    • Translation: RNA is translated into a polypeptide chain by ribosomes.
    • Folding: The polypeptide chain folds with the help of chaperones to form a functional protein.
    • Amino acids are the building blocks of proteins.

Studying Gene Function

  • Manipulate the gene of interest and observe the biological consequences.
    • Gain-of-Function: Over-expression of the gene of interest or a constitutively active mutant protein.
    • Loss-of-Function: Expression of a dominant-negative mutant protein or knocking down the gene of interest.
  • Assessment Methods: Numerous methods are available to assess the effects of gene manipulation.
  • Detection Methods: Various methods exist for protein and nucleotide detection.

Recombinant DNA Technology: Expression Vectors

  • Plasmids: Common vectors for cloning DNA inserts up to 4kb.
    • Promoter and Multiple Cloning Site (MCS): Contains numerous restriction endonuclease cleavage sites for DNA insert cloning.
    • Origin of Replication (ori): A DNA sequence that signals the host cell DNA polymerase to replicate the DNA molecule.
    • Antibiotic Resistance Gene (e.g., Ampicillin; Ampr): Used for selecting cells that have the plasmid.
  • Bacteriophages: Can be used for larger DNA fragments, such as genomic or cDNA, up to 100kb.
  • Idealized Plasmid Map: Illustrates the arrangement of key elements within a plasmid vector.

Cloning and Transformation

  • Cloning: Inserting a gene of interest into an expression vector.
  • Transformation: Introducing the recombinant vector into cells.
  • Producing Enough Vector Molecules: Essential for conducting studies that require sufficient amounts of the vector.

Delivery of Cloned Genes to Cells

  • Goal: To deliver the recombinant gene into cells for recombinant gene expression.
  • Delivery Strategies:
    • Transfection: Introducing genetic material (DNA) into cultured cells.
    • Electroporation: Creating transient pores in the cell membrane to allow DNA entry.
    • Virus-Mediated Transduction: Using viruses to deliver genes into cells.

Introducing Genetic Material into Cultured Cells

  • Introducing DNA into cultured cells so the cells can express the protein of interest.
  • Methods:
    • Calcium Phosphate Transfection
    • Liposomes (Lipofection)
    • Electroporation
    • Virus-Mediated Transduction

Protein Detection Methods

  • Confirming Protein Expression: After delivering the gene of interest into cells.
  • Methods:
    • Immunofluorescence Staining
    • Immunoblotting (Western Blot Analysis)

Antibodies for Protein Detection

  • Studies of gene expression and function often require the detection of proteins.
    • Ectopic expression of transgenes.
    • Upregulation/downregulation of downstream/endogenous genes.
  • Applications for detecting proteins (utilizing antibodies):
    • Immunoblotting
    • Immunoprecipitation
    • Immunofluorescence
  • Antibody Production:
    • Lab animals are injected with a peptide/protein.
    • B lymphocytes of the host organism's immune system raise antibodies to the foreign molecule.
    • Sera from immunized animals (e.g., mice, rabbits, goats, donkeys) are collected, and the antibodies raised (by B lymphocytes) are isolated for use in experiments.

Immunofluorescence Staining

  • Example: Rat cortical neurons and glia stained for Neurofilament-H, nucleus, cell membrane, and cytosol.

Reporter Molecules - Fluorophores

  • Use of fluorescent conjugates.
  • Examples:
    • AMCA
    • BV421
    • mHoneydew
    • YFP (Citrine)
    • EGFP
    • ECFP
    • OmOrange
    • mBanana
    • mGrape1
    • mRaspberry
  • Common Fluorophores and Their Properties:
    • Alexa Fluor 400
    • Alexa Fluor 554
    • R-PE
  • Example Image: Red (Actin), Green (Myosin), Blue (Microtubule)

Immunoblotting (Western Blotting)

  • Proteins in cell extracts are loaded onto an acrylamide matrix gel and electrophoresed.
  • Proteins separate by size: smaller proteins migrate faster than larger ones.
  • Proteins are transferred to a membrane, and antibodies are used to detect the proteins on the membrane.

Western Blotting Steps

  • A mixture of proteins is solubilized and separated by SDS-PAGE.
  • The proteins are then blotted onto a membrane using an electrical current.
  • The blot is washed, incubated with primary antibody, washed, and exposed to secondary antibody.
  • The secondary antibody is conjugated to an enzyme that can be detected through chemiluminescence.
  • Chemiluminescence: The generation of light through a chemical reaction.
  • Or fluorescent protein-conjugated secondary antibodies are used for detection.

Western Blotting Visualization

  • Protein of interest visualized by chemiluminescent Protein A.
  • Protein of interest visualized by fluorescent Protein A (Protein B also shown).

Western Blotting Applications

  • Cultured cells were treated with growth factor EGF.
  • Cell lysates were prepared, proteins separated by SDS-PAGE, then subjected to WB using antibodies against Erk and phosphorylated form of Erk (p-Erk).
  • Example: 0, 1, 3 mM EGF.

Information from Western Blot Results

  • Presence of proteins of interest.
  • Levels of protein expression.
  • Levels of protein activation (requires activation state-specific antibody).

Reporter Genes: Green Fluorescent Protein (GFP)

  • Aids in LIVE tracking of proteins in cells.
  • Your protein + GFP.