Transfection and Protein Localisation

8/14/2019 Transfection and Protein Localisation

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Transfection andProtein localization



Exploring protein function

1) Where is it localized in the cell?

2) What is it doing in the cell?

Approaches:a) Make antibodies - immunofluorescence

b) “Express” the protein in cells with a tag¥ Fuse to GFP

Approaches:a) Reduce protein levels - RNA interference

b) Increase protein levels “over-express”

c) “Express” mutant versions



Exploring protein function

1) Where is it localized in the cell?

2) What is it doing in the cell?

Approaches:a) Make antibodies - immunofluorescence

Approaches:a) Reduce protein levels - RNA interference

Transfection!!!!

b) “Express” the protein in cells with a tag¥ Fuse to GFP

b) Increase protein levels “over-express”

c) “Express” mutant versions



Transfection =

Introduction of DNA into mammalian cells

Gene is transcribed and translated into protein

= “expressed”



Direct introduction of the DNA

Electroporation - electric

field temporarily disruptsplasma membrane

Biolistics (gene gun)- fireDNA coated particles intocell

Microinjection



Infection:Use recombinant viruses to deliver DNA

RetrovirusesAdenoviruses

Virally-mediated introduction of the DNA



Positively charged carrier moleculesare mixed with the DNA and added tocell culture media:

Calcium Phosphate

DEAE Dextranliposomesmicelles

Carrier-DNA complexes bind to plasmamembrane and are taken up

Carrier-mediated introduction of the DNA



Types of Transfection

Transient:Expression assayed 24-48

hours post transfection

Stable:Integration of the transfected DNA

into the cell genome - selectable

marker like neomycin resistancerequired

“stably transfected” cell line



DNA “expression” vector transfected:

pCMV/GFP

C M V

P r o m

o t e r

Insert gene

in here

Polyadenylationsite

S V 4 0

P

r o m o t e r

N e o

m y c i n

r e s i s t a n c e

PolyadenylationsitepUC

Bacterial origin of replication

A m p

i c i l l i n

r e s i s

t a n c e

Forexpressionin cells

Togeneratestable cellline

Foramplificationof theplasmid inbacteria

G F P



PROTEIN X

PROTEIN Y

GFP

GFP

GFP ZPROTEIN

Three ways to make Green fluorescentprotein “GFP” fusion constructs:





Some Cellular Organelles



•Compartments/organelles examined•Protein sequences sufficient for localization•Vital stains

Secretory Pathway:Endoplasmic Reticulum

Golgi Complex

Endocytotic Pathway:Endosomes

Mitochondria

Nuclei



Transport through nuclearporesignal = basic amino acid

stretchesexample: P-P-K-K-K-R-K-V

Nucleus



Import of proteins into nucleus through nuclear pore



Nuclear Stain:

Hoechst 33258binds DNA



Transmembrane transport signal

Example: H2N-M-L-S-L-R -Q-S-I-R -F-F-K-

P-A-A-T-R -T-L-C-S-S-R-Y-L-L

Mitochondria



Protein being transported across mitochondrial membranes



Mitochondrial dye =MitoTracker Red

Non-fluorescent until

oxidized

Accumulates inmitochondria and oxidized

Diffuses throughmembranes

Mitotracker

DNA



nuclear envelope

endoplasmic reticulum

lysosome

earlyendosome

lateendosome

Golgi apparatus

cis

Golginetwork

trans

Golginetwork

Golgistack

CYTOSOL

plasmamembrane

Cellular components of the secretory and endocytic pathways



Entry into E.R.:Transmembrane transport signal= hydrophobic amino acid stretches

Example: H2N-M-M-S-F-V-S-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-Q

Retention in E.R. lumen:

Signal = K-D-E-L-COOH

Endoplasmic Reticulum

at amino terminus

at carboxy terminus





Mitotracker Red and ER-blue/white



Golginucleus

From the ER, secreted and membrane proteins move to the Golgi, aseries of membrane-bound compartments found near the nucleus



BODIPY-TR ceramide

Golgi marker

Ceramide = lipid

When metabolized,concentrates in theGolgi

Red fluorophore





Golgi (ceramide)

Lysosomes (LysoTracker)

DNA (Hoechst)

MDCK CellsMadin-Darby Canine Kidney

Polarized Epithelial Cells

Molecular Probes, Inc.







Endosomes - pinch off from plasma membrane

Clathrin -coated pits and vesicles



RECEPTOR-MEDIATED ENDOCYTOSIS occurs through specialmembrane sites coated with the protein CLATHRIN.Receptors interact with clathrin indirectly, through

ADAPTIN proteins.Coated membrane buds that contain clathrin, adaptins, and

receptors bound to their ligands pinch off to form coatedvesicles.



Iron is carried inblood by the protein

TRANSFERRINand is taken up intocells by endocytosismediated by the

TRANSFERRINRECEPTOR

Inside the endosome

Fe3+

is released.Transferrin receptorsthen return to thecell surface, wherethe transferrindissociates



Rhodamine transferrin

Does the fluorescent green protein co-localize?



TODAY:•Transfect Cells transiently with unknownprotein X, Y or Z fused to GFP

In two days:

•Vital stain with another dye to compare•Visualize both GFP and dye in the sameliving cells! by fluorescence microscopy

Where are the unknown proteinslocalized???

Documents

Transfection and Protein Localisation