L15 - Visualising gene expression

what does gene expression determine

cell identity

what is one way to study genes?

Muatate genes and see what effect it has on phenotype

e.g. fruit fly muatation leads to change in body structure

what can we use to detect proetins

use antibody if Ab binds that means proetin is present

how are Ab made

• inject proetin into mouse

• get immune reposnse

• remive blood and collect B celss in blood

• combine with tumour cells

• form a hybridoma

• makes Monoclonan Ab

visulalising Ab staining process

• fix cells

• perminabalise membranes

• add Ab

• then wash asway an Ab taht did not bind

• then add 2nd Ab comp to Ab with a label on it

• visualluy see labelled secondary Ab

2nd Ab could be enzyme or flurecently labelled

can same Ab be used in different species

yes gene called distalless involved in appendages

• gene was foudn in multiple different species shwoing how its conserved

different ways can label Ab

fuorophore tagged Ab (absirbe one color and emmit another ) e.g. Texas red → use florescent microscope

• didfern florophore emit differnet colours so can corosonp each colour witha diff gene/ protein

enzyme tagged Ab → Horse radish peroxidase and alkali phosphitase catalyse reactions that cause colored percipitate

Other ways to detect proetin

using phalloidin → from mushroom

binds to actin whihc is in muscles cells and prevents depoluymerization (actin doesnt break apart) and so stains it

what is in situ hybridization used for and how how dies it work

• used to visualise mRNA levles - so tracsription of genes

• fixed a permiabilised cells

• add antisese probe and high temp

• wahs out any unannealed antisense probe

• visualise remiaining probe

How do welabel the mRNA probes

• using haptens - that are recognised by Ab

• e.g. Biotin in mRNA, fluorescent labled streptavin binds to biotin

• e.g. Hapten is bound to by enzyme encouperated Ab enzyme makes a percipitate appear

• also label using radioactive Pi so can be visulaed using autoradiography

what is WMISH

Whole Multiple In Situ Hybridization

• mutiple probes used able to decet genes simultaniously over the body

HCR

• amplify signals of In situ hybridization

• as some times not much mRNA so fluoresent is drowned out:

• Fix and permeabilise cells/tissue

• Add probes complementary to target mRNA

• Probe binds target mRNA by complementary base pairing

• Bound probe exposes initiator sequence

• Add fluorescent H1 and H2 hairpins

• Initiator opens H1 hairpin

• Open H1 exposes sequence that opens H2

• Open H2 opens another H1

• Chain reaction continues forming long fluorescent chain

• Many fluorophores accumulate at one mRNA site giving strong signal

chromosomal FISH

shows order of genes on chromosome

how are reporter genes used

• Help detect gene expression

• repance coding area of DNA with reporter gene so when gene is terned on reporter e.g. GFP from jelly fish - glow grenn when switched on