Basic principles and techniques Flashcards
What techniques can be used to see when and where the gene is expressed in the embryo
In situ hybridization, Northern blot, RT-PCR, Micro-array, Reporter lines (transgenes)
Outline the method of in situ hybridization
Produce a DNA or RNA probe that is complementary to the mRNA being transcribed in the cell
Have the probe labelled with a radioactive isotope or fluorescent tag or enzyme.
Radioactive labels are detected by radiography
Fluorescent tags are detected by fluorescent microscopy
Enzyme labels are detected by the enzymes conversion of a colourless substrate into a coloured product.
How would a researcher discover if the protein is expressed at the same time as the gene.
Immunohistochemistry
Produce antibodies specific for the protein of interest
The antibody can be tagged directly with a coloured dye or an enzyme (HRP)
What is the difference between western and northern blotting
Northern blotting = mRNA detection
Western blotting = Protein detection
How does western blotting work
Collect sample of cells to be investigated
Lysis of cells by detergents, protease inhibitors to prevent digestion of proteins
Blend the cells and separate compartments by centrifugation
SDS-PAGE to separate proteins based on size
Transfer onto membrane and add primary antibody/enzyme/fluorescent tag
Wash - Detection
How does a microarray work
A collection of DNA spots are present on a solid fixed surface, These are used to hybridize cDNA/RNA which has a fluorescent probe attached.
Why would you use RT-PCR over western blotting
When you have isolated your sample RNA from a very small sample of cells (ie the wing disc of drosophila) Won’t produce significant enough levels for use in western blotting
What is transgenesis
Addition of an extra copy of DNA in to an animal model - so can be applied to all genetic models (drosophila, mouse, zebrafish)
Replace the gene of interest with GFP after regulatory sequence of gene of interest. Whenever the gene would normally be expressed GFP is produced instead visualizing where it would normally be expressed.
Problems with transgenesis
Random insertion of foreign DNA into the host genome. Areas of the genome of the host will be non-favourable (highly condensed areas of chromatin)
Piece of DNA inserts in the middle of another gene creating a mutation
How does the mouse overcome the problems associated with transgenesis
Knock ins
Place the reporter gene precisely in the locus of the gene of interest
If the dosage of a gene is nonessential for life - in one copy of the gene remove the gene and replace it with GFP
Mice have one copy expressing the gene and one expressing GFP
How do you produce a knockout mouse
Produce an inactive copy of the gene of interest which contains some drug resistance.
ES cells are introduced to the construct by transfection.
Homologous recombination can then occur creating an inactivated copy of the gene with some drug resistance. Grow cells in culture and select for drug resistance. Inject cells into inner cell mass of blastocyst
Chimeric animal produces gametes derived from stem cells
Breed chimeras and resultant mice may have knockout present in all cells
How to ensure the gene downstream of GFP is in frame at translation
Place the start site of GFP precisely where the start site of the gene of interest and then insert the sequence that codes for GFP
Most of the time this disrupts the reading frame of the gene of interest at the point of translation.
