Determining gene expression and gene function Flashcards
(17 cards)
how can we find out if a gene is sufficient?
a gain of function approach
place the gene product somewhere else - out of context and then essay for the deregulation of the downstream events
what is the embryological equivalent to aa gain o function approach
grafting =
pasting
grafting of cells or tissues
Mangold and Spemann grafting of the organiser
- homotopic and isochronic (replacing like with like) for fate mapping
e. g. zone of polarising activity from the caudal or posterior margin of a chicken in bud was graft to the rostral or anterior margin = mirror image duplication of the digits - heterotopic = to study the effect of e.g.a signalling centre
homo topic grafting
grafting cells to the very site where they had been in the donor
tissue can be replaced by its exact counterpart but from a donor animal whose cells are labelled
= to trace where cells went to and which organs they contribute to
isochronic
exchanging cells at the same time of development
heterotophic
to a new site
e.g. grafting of an additional notochord sowing induction of the floor plate
heterochronic
to a new host w a diff age
implantation of the immatuure segmental plate instead of sorties
to see if paraxial mesoderm has to mature and produce somites before it can induce maturation of neural tube and initiation of neurogenesis
reverse genetics
you go from the gene to the phenotype that arises from your genetic manipulation
approaches to test gene function by making intervention and then assaying for phenotypes that are divergent from the norm
forward genetics
mutagenesis screens when screening by phenotype
from phenotype to gene that had been manipulated
observation
hypoethsis
experiment
conc
myod expressed before myog
myod - myog
myod & myod misexpressed
myog expression deregulated?
detection method to visuals gene exp
painting
painting - when n where gene is expressed
fate mapping
embryological/anatomical
labelling cells by injection of fluorescent dye + fate following
observation of cross morphlogy
histology
tracin of scientific structures
electron.transmission microscope scanning = resolution
fluorescence microscope
confocal scanning
molecular way
differential gene expression
detect mRNA or protein for gene expression
cutting
ablation
to study inductive interaction
loss of function
necessary
pasting
gain of function
sufficient
methods to detect gene expression
mRJNA + protein can be detected insitu at the anatomical location where the gene is expressed
- mRNA = hybridising it with a labelled antisense RNA molecule
- protein = immunohistochemistry
to quantify how much mRNA or protein is expressed
biochemical methods needed
- extraction required
- mRNA
run extracted RNA on a gel gel, transfer content of the gel onto a filter + hybridise with an antisense probe
= northern blot - reverse transcription followed by PCR = RT-PC R run out PCR product to estimate how intense the bands are on the gel
quantise method = Q-PCR - microarray or RNA-SEQ
cyber green quantitative par of turckermans
the more fluorescent the more upwards is the par product
more per products the more RNA was there
in proteins”
1, western blot
run a protein extract on a denaturing gel. transfer into filter
apply enzyme coupped antibodies
offer enzyme suitable substrate
measure enzymatic conversion of the substrate
- ELISA - enzyme linked immune solvent assay
proteins immobilised in micro plates
protein preparations added to cover all the unsaturated surface binding sites on the plate
specific antibody that recognises the protein were after is added
reporter enzyme coupled
allows to quantify how much protein is there in the first place
immuno histochemsitry or inset hybridisation
ISH
exploits the base paring of nucleic acids
in vitro genetrate a anitisense so a paralelel complemntary labelled probe
this nucleotides are labelled w DIG deoxygenated a steroid
antibody that recognises DIG
therefore the antibody will bind to the messenger RNA probe hybrids
antibody is coupled to alkaline phosphatase enzyme
when substrate is added + alkaline PH
colour precipitate is generated wherever the RNA has been made
immuno
antibody detects particular protein of interest
even post translation modifications
antibody is couples to an enzyme or fluorochrome
but usually secondary antibody used to the enhance signal
if first antibody was generated in a mouse by immunising the mouse with the original protein
he mouse’s immune response and isolating the antibody from the mouse blood
you will have an antibody where the variable part of the antibody will be specific for your protein
he constant part would be that of a mouse immunoglobulin
you would now introduce an anti mouse antibody as a secondary antibody.
an anti mouse antibody made in a goat.
its variable part would be specifically detecting the mouse immunoglobulins
It’s constant parred would be copied to an enzyme that is in in immunohistochemistry or it would be copied to a floral crom
If you had an enzyme kopit secondary antibody and this may even be again alkaline phosphatase,
you create a coloured precipitate in the same way as in the in situ hybridisation
immune histochemsitry or inset hybridisation
In an in C two, you did take messenger RNA with the complimentary RNA probe
the probe is detected with an antibody directed against the label of the probe.
in immunohistochemistry or immuno fluorescence you detect a specific antigen. Typically a protein with a primary antibody.
the primary antibody is detected with a secondary antibody.
transgene constructs
a gene newly transferred into cells
indoginous
normally in the cells
