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.