Transcript Selectable marker For mammalian cells

Lab 5A and 5B Overview
This Week
Last Week
Investigating protein sorting signals using cloning, transfection,
GFP-fusion proteins, and vital stains for cellular compartments
1. Protein sorting and membrane trafficking
- or How cells deliver things to the right place
2. Fluorescent proteins are critical tools in
Cell biology
3. Transfection and transgene expression
- or How we get DNA into cells
to express “designer” genes
4. Fluorescent markers for different
compartments of the secretory
and endocytic pathways
Fusion proteins are usually introduced into cells as DNA constructs
Protein X
GFP
Prote
pCMV:
Strong, constitutive
promoter
Ampicillin:
Selectable marker
for bacterial cells
GFP
Protein Y
GFP
in Z
BGH pA:
Polyadenylation
sequence
Neomycin:
Selectable marker for
mammalian cells
pUC:
Origin of replication
for bacterial cells
Cloning vector for expressing GFP fusion proteins
in mammalian cells (constructed in bacteria)
DNA is a large, charged molecule that normally doesn’t cross
cell membranes… so we have to use tricks to get it into cells
TRANSFECTION - from trans, meaning “across”
The negatively-charged phosphate backbone of DNA
must be neutralized by positively charged counterions
to allow transport across the plasma membrane.
Effectene
®
Outline of transfection
protocol (Qiagen)
Principle of transfection
with Effectene® reagent
+ Enhancer
There are many points along the way where
transfection efficiency can be compromised
Proton sponge hypothesis:
sequestration of cations by
DNA leads to the osmotic
swelling and rupture of
endosomes, releasing DNA
vector into the cytoplasm
Only a fraction of treated cells will be successfully
transfected, and thus the expression of the transgene
will be quite VARIABLE - here GFP is used as a marker
of transfection
Fusion proteins are usually introduced into cells as DNA constructs
Protein X
GFP
Prote
pCMV:
Strong, constitutive
promoter
Ampicillin:
Selectable marker
for bacterial cells
GFP
Protein Y
GFP
in Z
BGH pA:
Polyadenylation
sequence
Neomycin Resistance
Gene: Selectable marker
For mammalian cells
pUC:
Origin of replication
for bacterial cells
Cloning vector for expressing GFP fusion proteins
in mammalian cells (constructed in bacteria)
Alternative strategies to ectopically express genes/siRNAs
Electroporation
Retroviral Vectors
Transfect
-High Efficiency
-Stable DNA integration
-Replication Incompetent
-Level 2 Bio-Safety
Retrovirus
-High Efficiency
-Many Cells/even intact tissues
-Cell Fusion
-Loss of intracellular components
Alternative strategies to ectopically express genes/siRNAs
Gene Gun
-Applicable to many tissues
-Penetrates Mitochondria/Chloroplasts
-Shallow penetration of particles
-Cell damage
Microinjection
-No selection process
-DNA delivery accurately controlled
-Minimal perturbation of cells
-Technically difficult/few cells
4 transfected
constructs
U
X
Y
Z
5 vital dye
counterstains
Golgi
Endosome
Mitochondria
ER
Nucleus
Ceramide is a LIPID that gets trapped after modification
in the Golgi apparatus
BODIPY®-TR Ceramide (Molecular Probes)
Fluorophore
Label
Ex
Em
BODIPYTR
Green
Red
Cultured Epithelial Cells
DNA (Hoechst)
Golgi (ceramide)
Steve Rogers, U. Illinois
Iron is carried in
blood by the protein
TRANSFERRIN
and is taken up into
cells by endocytosis
mediated by the
TRANSFERRIN
RECEPTOR.
Rhodamine-labeled
TRANSFERRIN protein
can be used to track
receptor-mediated
endocytosis
EX
Green
EM
Red
MitoTracker Red CM-H2XRos
“…the reduced versions of these probes do not fluoresce
EX
Green
EM
Red
until they enter an actively respiring cell,
where they are oxidized to the fluorescent
mitochondrion-selective probe and then
sequestered in the mitochondria.”
MOLECULAR PROBES handbook
Cultured Lung Epithelial Cells
DNA (DAPI)
Mitochondria (MitoTracker)
Actin (Phalloidin)
Image from Nikon
ER-Tracker Blue-White DPX
“ER-Tracker
Blue-White DPX is a highly selective and
photostable stain for the ER in live cells…
Staining at low concentrations does not appear to be toxic to cells.”
(MOLECULAR PROBES Handbook)
EX
UV
EM
Blue
Cultured Endothelial Cells
ER (ER-Tracker)
Image from Invitrogen
4 transfected
constructs
U
X
Y
Z
5 vital dye
counterstains
Golgi
Endosome
Mitochondria
ER
Nucleus
20 Data
Sets
Potential Challenges Encountered
in this Week’s Lab
Data Management
Autofluorescence - increases as cells die
Bleed-through between different filter sets
Nonspecific labeling of organelles
Photobleaching
Fluorescence Microscopy
Stokes’ shift
em
intensity
ex
wavelength
excitation
and emission
filters
Fluorophore
(or “Fluorochrome”)
Excitation
maximum
Emission
maximum
Fluorescein
490
520
Rhodamine
550
580
DAPI or Hoechst
345
455
Fluorescence wavelength filters must be designed to match the
excitation/emission spectra of the fluorophores you plan to use.
Red Channel
Bodipy-TR ceramide
GFP Channel
Autofluorescence
of dying cells
GFP
Simultaneous localization of cellular components
To be useful, a “counterstain”
should fluoresce at a wavelength
different from GFP - e.g. RED or BLUE
N
-Invitrogen
Mitochondria (MitoTracker)
Lysosomes (Lyso-Tracker)
“Colocalization” can help to
establish that two
molecules are in the same
place at the same time.
If the location of one is
known, it can reveal the
location of a less wellcharacterized component.
GFP Fusion
Protein
Mitochondria
(MitoTracker)
Colocalization
-Beech et al. Science (2000)