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Lecture 1: Cell and Tissue Culture > Lecture 1b - Transfection > Flashcards

Lecture 1b - Transfection Flashcards

1
Q

Transfection is the ________ into ________

A

delivery of DNA into eukaryotic cells

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2
Q

Transfection
Genese can be expressed either:
-
-

A

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

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3
Q

What are 3 broad reasons of why we transfect?

A

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

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4
Q

Dominant-negative

A

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

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5
Q

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

A

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

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6
Q

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

A

Promoter
MCS
PolyAsite
Ori
Antibiotic selection

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7
Q

What are the advantages of GFPs?

A

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

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8
Q

What are the disadvantages of GFP?

A

large protein tag
not great for bcem

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9
Q

What are some other epitope tags?

A

HA tags (influenza hemaglutinin)
Myc tags
FLAG

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10
Q

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

A

adherent cells
transient or stable

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11
Q

What is the mech of calcium phosphate?

A

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

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12
Q

benefits of CP

A

Low toxicity of reagent, inexpensive

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13
Q

drawbacks

A

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

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14
Q

DEAE-Dextran
Primarily used for transfection ____ cells

A

COS cells derived from monkey kidney tissue

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15
Q

mechanism of DEAE

A

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

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16
Q

Benefits and drawbacks of DEAE-Dextran

A

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

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17
Q

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

A
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18
Q

What is the mech of lipofection?

A

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

19
Q

What are the benefits and drawbacks of lipofection?

A

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

20
Q

What are the 4 major steps to generate transient transfectants?

A

Prepare DNA
Prepare cells
Prepare transfection reagent
Transfect

21
Q

Prepare DNA to transfect

A

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

22
Q

Prepare cells

A

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

23
Q

Prepare transfection reagent

A

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

24
Q

Transfect

A

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

25
Q

Benefits and drawbacks of immunofluorescence microscopy

A

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

26
Q

Western blotting/ELISA pros and cons

A

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

27
Q

Functional assays such as ____ or _____ are advantageous because

A

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

28
Q

Does the transfection eff increase with increase amount of DNA

A

not always

29
Q

Negative and positive controls

A

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

30
Q

What are 3 factors for transfection efficiency?

A

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

31
Q

Why are transients transient?

A

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

32
Q

How are stable transfectants different than transient?

A

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

33
Q

What must we do for stable transfectants? 2

A

-select and “clone” transfectants
-screen for expression of gene of interest

34
Q

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

A

Transfection - lipofection
Selection - drug resistance

35
Q

Selection markers
Antibiotics/drug sensitivity
Give 2 examples

A

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

36
Q

Steps to constructing a stable cell line

A

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

37
Q

Why do we want to regulate gene expression?

A

some genes are toxic when expressed in mammalian cells

38
Q

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

A

Inducible promoters
Repression/de-repression

39
Q

Inducible promoters general idea

A

Transcription is at low basal levels in absence of inducer

40
Q

What is an example of an inducible promoter?

A

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

41
Q

Tet expression systems depend on the activity of an ________

A

inducible transcriptional activator, TA

42
Q

In the Tet-off system transcription …

A

is inactive in the presence of Tc or Dox

43
Q

In the Tet-on system transcription is …

A

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

44
Q

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

A

TRE

Lecture 1: Cell and Tissue Culture (5 decks)

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