Lecture 1b - Transfection

Question 1

Transfection is the ________ into ________

Answer 1

delivery of DNA into eukaryotic cells

Question 2

Transfection
Genese can be expressed either:
-
-

Answer 2

Transiently - 1-4 days
Stably - generation of a cell line perm expressing gene of interest

Question 3

What are 3 broad reasons of why we transfect?

Answer 3

Study gene of interest
-express mutant alleles of gene of interest
Ectopic expression of proteins
-express genes where they’ve never been before
Protein production

Question 4

Dominant-negative

Answer 4

non-functional allele that out-competes wild-type, creating a cell line def in gene of interest

Question 5

What is usually used for mammalian genes? What is cloned into the vector?

Answer 5

cDNAs - comp DNA to mRNA
Only the coding region (ATG….TAA)

Question 6

What are the 5 things included into vectors for mammalian gene expression?

Answer 6

Promoter
MCS
PolyAsite
Ori
Antibiotic selection

Question 7

What are the advantages of GFPs?

Answer 7

Intrinsically fluorescent so no labelling required - can work in live or fixed cells and can label with multiple colours
Fluorescence is stable

Question 8

What are the disadvantages of GFP?

Answer 8

large protein tag
not great for bcem

Question 9

What are some other epitope tags?

Answer 9

HA tags (influenza hemaglutinin)
Myc tags
FLAG

Question 10

Calcium phosphate
Primarily used in _____ cells
Can be used to construct either ____ or _____ cell lines

Answer 10

adherent cells
transient or stable

Question 11

What is the mech of calcium phosphate?

Answer 11

Calcium phosphate forms an insoluble precipitate with DNA which attaches to cell membrane and is endocytosed

Question 12

benefits of CP

Answer 12

Low toxicity of reagent, inexpensive

Question 13

drawbacks

Answer 13

reagent can be tricky to prepare, low efficiency in some cell types (cells need to be able to acitvely endocytose)

Question 14

DEAE-Dextran
Primarily used for transfection ____ cells

Answer 14

COS cells derived from monkey kidney tissue

Question 15

mechanism of DEAE

Answer 15

Positively charged DEAE-dextran binds to phosphate groups on DNA forming a precipitate which is endocytosed

Question 16

Benefits and drawbacks of DEAE-Dextran

Answer 16

Optimized for COS cell transfection and inexpensive
Only useful in a limited number of cell types

Question 17

Lipofection can work in both cell lines and suspension and adherent cells and can be used to generate stable and transient cell lines.

Question 18

What is the mech of lipofection?

Answer 18

Cationic lipids are used to create artificial vesicles (liposomes) that bind DNA molecules. The liposomes adhere and fuse with negatively charged membrane promoting DNA uptake

Question 19

What are the benefits and drawbacks of lipofection?

Answer 19

PROS
High eff, works in a variety of cell types

Drawbacks
Cost, cell type specificity, conditions must be optimized for efficiency and cytotoxicity, must be done in serum free media

Question 20

What are the 4 major steps to generate transient transfectants?

Answer 20

Prepare DNA
Prepare cells
Prepare transfection reagent
Transfect

Question 21

Prepare DNA to transfect

Answer 21

DNA must be very pure, Qiagen column or 2X-CsCL gradient

Question 22

Prepare cells

Answer 22

adherent cells split the night before to 60% confluence
Change to appropriate media

Question 23

Prepare transfection reagent

Answer 23

Mix DNA with reagent to form precipiate or liposomes
May need to optimize DNA:reagent ratio

Question 24

Transfect

Answer 24

Change media on cells if needed
Add reagent-DNA mix to cells. Remove after 4-8hrs
Culture cells for 12-96hrs

Question 25

Benefits and drawbacks of immunofluorescence microscopy

Answer 25

benefits -can examine gene expression in indv cells -very visual, can see where protein localizes/colocalizes drawbacks -does not necessarily determine whether gene product is functional -low throughput

Question 26

Western blotting/ELISA pros and cons

Answer 26

pros high thorughput cons hard to detect protein of interest if the transfection eff is low

Question 27

Functional assays such as ____ or _____ are advantageous because

Answer 27

ligand binding, electophysiology because we can tel if expressed gene product is functional

Question 28

Does the transfection eff increase with increase amount of DNA

Answer 28

not always

Question 29

Negative and positive controls

Answer 29

Negative -"random" DNA -controls for toxicity of transfection system so reagent alone or procedure alone Positive -Transfect cells with vector alone

Question 30

What are 3 factors for transfection efficiency?

Answer 30

Technical factors -impure DNA -cells too confluent, old Cell related issues -cell type difficult to transfect Gene toxicity -expression of gene is toxic to cells

Question 31

Why are transients transient?

Answer 31

Mammalian expression plasmid does not replicate in most cells so plasmid gets "lost" as cells divide

Question 32

How are stable transfectants different than transient?

Answer 32

stable - DNA integrated into mammalian genome selection pressure keeps gene of interest integrated into genome

Question 33

What must we do for stable transfectants? 2

Answer 33

-select and "clone" transfectants -screen for expression of gene of interest

Question 34

What 2 methods are used for generating a stable cell line?

Answer 34

Transfection - lipofection Selection - drug resistance

Question 35

Selection markers Antibiotics/drug sensitivity Give 2 examples

Answer 35

G418- aminoglycoside antibiotic - blocks protein synthesis Hygromycin B- aminocyclitol antibiotic, inhibits protein synthesis

Question 36

Steps to constructing a stable cell line

Answer 36

Limiting dilution cloning -24hrs post transfection, split cells into selective media The transfected cells expressing resistance gene will grow, others will die Pick isolated clonies, culture in indv wells of multi-well plate Detection if cells express gene, all cells in well should do so, can use higher throughput methods Subcloning Cells may still be genetically unstable so need to reclone by limiting dilution for any good candidates

Question 37

Why do we want to regulate gene expression?

Answer 37

some genes are toxic when expressed in mammalian cells

Question 38

What are the two types of systems to regualte gene expression?

Answer 38

Inducible promoters Repression/de-repression

Question 39

Inducible promoters general idea

Answer 39

Transcription is at low basal levels in absence of inducer

Question 40

What is an example of an inducible promoter?

Answer 40

Mouse mammary tumor virus promoter is low in the absnece of DEX. addition of DEX enhances gene expression

Question 41

Tet expression systems depend on the activity of an ________

Answer 41

inducible transcriptional activator, TA

Question 42

In the Tet-off system transcription ...

Answer 42

is inactive in the presence of Tc or Dox

Question 43

In the Tet-on system transcription is ...

Answer 43

active in the presence of DOX (responds poorly to Tc)

Question 44

In the presence of Dox or Tc rTA is not bound to

Answer 44

TRE