QP-Gene Reporter Assays

what is the general principle of cell signalling pathways? 6

• ligand-receptor binding

• activation of signalling proteins

• signalling proteins can be activated by phosphorylation- make docking sites for other signalling proteins

• recruitment and activation of downstream proteins

• TF activation: eneters/acts in the nucleues

• gene expression

what can reporter assays used to determine? 3

• determine where a signalling molecule is situated in a signalling pathway

whether a sigalling pathway or TF is activated

if signalling molecule can activate a TF

what can TLRs identify? 4 and what do they do?

• flagellin

• LPS

• dsRNA

• ssRNA

detect PAMPs- TLR—>adapter proteins→kinases/signalling proteins→NKkB→gene expression

TLR signalling pathway for TLR4 7

• TLR dimerisation

• adaptor recruitment

• Myd88

• IRAK4/1

• TRAG6

• IkB degradation

• NFKB

how does IkB function? what inflammatory genes can NFKB control?

IkB inhibita NfKB

• once activated

• IkB is phosphrylated- tagging it for ubiquitinaation-IkB degradation- NFKB to the nucleus realease

• iNOS

• COX2

• Cytokines

what does Mal do?

• Mal plays a role in TLR4 signalling

• interacts with TLR2 and Myd88- from protein protein studoes

• knockout studies- Mal signals through TLR4, TLR2

how would you test if a sgnalling molecule activates a TF?

• overexpress the signalling molecule and test if it activates the TF

• IE overexpress Mal and emasure NFKB activity

• 
  

how do overexpression vectors work?

• in a mammalian expression vector

• mal cDNA→mammalian expression vector

• vector has a CMV promotor constitutive promotor-→restrction sites→Mal cDNA is under the control of the promotor

• Mal cDNA is in a constitutive promotr→gene is always expressed→overexpression

• he Insert (cDNA): This is the complementary DNA copy of the mRNA encoding your signaling protein. It contains only the coding sequence (exons) without introns.

• The Backbone (Expression Vector): This plasmid must contain a strong mammalian promoter (like CMV or EF1a) to drive high levels of transcription, a polyadenylation signal (like SV40 polyA), and a selectable marker (like Neomycin or Puromycin) if you plan to make stable cell lines.

• Methods: This is typically done using restriction enzyme cloning, Gibson Assembly, or Gateway cloning.

how are vectors intrduced in mammalian cells? what is a common cell?

transfection- reagent that punches holes to the cell membrane to allow DNA entry

• HEK293→easy to transfect, handle, cultre, supports cellular machinery for signalling

• can signal through many pathways

examples of reporter genes? how do they work? an example

1. luciferase

• NFkB activation→NFkB binds promotor→luciferase gene expressed→luciferase enzyme made→light signal

• light provides a measurable readout

how do dual reporter luciferase experimets work? how are they calculated

• 2lights

• firefly luciferase- experimental- under the control of promotor of interest

• renilla luciferaee- control- constitution promotor

• normalised actvity: firefly/renilla

• co-transfect firefly report and constitutive renilla reort- measure firefly and quench firefly- initiate renilla- luminometer

what is a dominant negative signalling protein? how does it map a patwhay?

mutated signalling protein that interacts with normal/WT protein to reduce signalling

• DN X blocks signalling of Y- X is downstream of Y

• if Y passes DN X Y is downstream of X

what is SEAP? why is it useful?

secreted embryonic alkaline phosphatase

• secreted into culture medium- no need for cell lysing and reorter activity can be measures uding fluorescent substrates

what is promotor bashing?

• promotor deletions and mutations- to see what is needed for gene expression

• clone different lengths upstream of reporter gene and introduce it

• if reproter actvity stops- region has important element

what is SNP analysis for?

tests whether a signal base change- cloned upstream of reporter gene- changes promotor/regulator region

main applications of reporter gene assays

• check promotor analysis- promotor bashing

• SNP analysis

• studying signalling pathways

• determine pathway order

• testing drugs that affect pathways