TB1-3: Getting DNA into cells Flashcards
What broad type of vector should be used for getting small DNA into bacteria?
Plasmids
What two methods are used to get small DNA (in plasmid vectors) into bacteria?
Chemical/Heat Shock Transformation
or Electroporation
What broad type of vector should be used for getting larger DNA into bacteria?
Cosmids or Fosmids
What method is used to get larger DNA (in cosmid or fosmid vectors) into bacteria?
Phage-mediated methods via in vivo packaging
Bacteria take up DNA naturally, so what’s the issue?
They take it up inefficiently
Describe the 6 key steps in bacterial transformation for plasmid take-up.
- add RbCl or CaCl2
- incubate on ice
- add the DNA
- Heat shock 42oC 10s-1min
- recovery
- add antibiotic
What is another name given to the pores created in the cell membrane of E.coli during rapid growth?
adhesion zones
Why can DNA not pass through the adhesion zones even though the zones are physically large enough?
Neg phosphates in both the cell membrane lipids and the DNA backbone
(electrostatic repulsion)
Why add RbCl or CaCl2 during bacterial transformation?
Ca2+ or Rb+ ions interact with the negative charges of the phosphates in the DNA backbone and lipid membrane to create electrostatic neutrality
Why incubate on ice in bacterial transformation?
- decreases the temperature
- causes a decrease in membrane fluidity and movement
- this stabilises the neg phosphates and makes them easier to be shielded ionically by Rb+ or Ca2+ ions
What temperature is the sample increased to during heat shock stage of bacterial transformation?
42oC
What is the purpose of the heat shock stage in bacterial transformation?
brief but rapid increase in temperature causes a temperature imbalance across different sides of the cell memrbane
- this creates a current and allows DNA to pass through the adhesion zones
what is the name given to a cell that can take up DNA? i.e. through its adhesion zones in bacterial cells?
competent cells
What is the basis of electroporation for getting DNA into bacterial cells?
a very high localised current between electrodes creates transient pores within the bacterial membrane
- the ‘electrophoresis-type effect’ enables negatively charged DNA to pass through
What temperature is electroporation carried out at? Why?
0oC becuase the high voltage used causes an increase in heat
therefore colder conditions are required to minimise cellular heat damage
How do you make E. coli electro-competent for use in electroporation?
wash it to remove salts
because salts in e.g., buffers conduct electricity
Name 6 comparative points between the chemical/heat shock method (H) of getting DNA into bacteria and the electroporation method (E)
- (C) no specialised equip. (E) expensive equip and consumables e.g. cuvettes
- (C) longer because incubation, (E) quick
- (C) DNA vol. not critical. (E) low vol. of DNA
- (C) tolerates salt in DNA preparation, (E) very sensitive to salt (hence the new cuvettes
- (C) lower transformation efficiency, (E) higher
- (C) generally reliable outcomes, (E) variable outcomes but can be excellent
Name the two common types of bacteriophage used for getting DNA into bacterial cells.
cosmids and fosmids
Why are bacteriophage used for getting DNA into bacterial cells?
they are a natural infection agent for bacteria and package DNA into phage heads
What size genomic restriction fragments of DNA do bacteriophage (cosmids/fosmids) take up?
35-45kb
Where is the cosmid vector cut with restriciton enzymes in order to insert the genomic restriction fragment?
polylinker
After the genomic restriction fragment is ligated to the cosmid vector in bacteriophage infection (getting DNA into bacteria cells), what happens?
there is in vitro packaging
-insert DNA is between adjacent COS sites
- causes lamda heads ak.a recombinant cosmid virions to be produced
How is selection for bacteria cells that have been infected by bacteriophage (in the process of getting DNA into cells) achieved?
selection with ampicillin
List 5 limitations of using bacterial hosts for making eukaryotic proteins.
1.codon useage differences
2. protein folding issues
3. targetting to mem, or other sub-cellular compartements not possible
4. no Ser/Thr/Tyr phosphorylation
5. no glycosylation
What is the possible result of codon usage differences between using bacterial hosts for making eukaryotic proteins? How can this be overcome?
Could cause premature chain termination
Overcome via Rosetta strain cells
How can possible protein folding issues from using bacterial hosts for making eukaryotic proteins be overcome?
Using Origami strains of cells
Why would we not use mammalian cell culture in our practicals?
They are difficult to work with e.g. need sterile conditions and specialised cells etc.
Why would one want to use mammalian cell culture in experiments? (4 reasons)
- to understand cell biology
- in the manufacture of proteins
- to test drugs and therapies
- for regenerative medicine
What is regenerative medicine?
“process of replacing, engineering or regenerating human or animal cells, tissues or organs to restore or establish normal function”
What sort of therapies would regenerative medicine include? (2 examples)
- skin grafts
- artificial livers
Give 3 examples of mammalian cells used in the lab and the tissue that they are isolated from.
- fribroblasts from skin
- hepatocytes from liver
- adipocytes from fat tissue
What are ‘organoids’?
tiny, self-organised 3D tissue cultures derived from stem cells. (Used as an alternative mammalian cell culture)
What are the conditions for the humidified incubator used in order to grow/keep alive mammalian cell culture?
37oC
5% CO2
2-20% O2
What is DMEM? What does it stand for?
Dulbecco’s Modified Eagle Medium
a medium often used to grow mammalian cell culture
Give 4 examples of nutrients and growth factors that must be in mammalian cell culture medium?
pyruvate
glucose
glutamate
FCS (fetal calf serum)
How regularly must you refeed or harvest mammalian cell culture medium?
Regularly i.e. ~ 2-3 days
Why must you refeed and harvest mammalian cell culture regularly?
the primary cells stop dividing when they touch
a.k.a contact inhibition
What broad type of mammalian cells don’t experience contact inhibition?
cancer cells
How can one monitor the proliferation of mammalian cell culture?
- live cell imaging
- or cell counting
What are primary (mammalian) cells?
cells that have been isolated directly from tissues
Describe the growth capacity of primary mammalian cells.
- they have a finite growth capacity
- when the max (Hayflick limit) is reached they then go into senescence
- can only divide ~40-60 times
DO primary cells need growth factors and a solid support?
yes
describe the contact inhibition that takes place between primary mammalian cells?
form a monolayer
- covers the surface
- stop growing when cells touch
What type of mammalian cell is a skin fribroblast when isolated for cell culture?
primary cell
What are immortalised (mammalian) cells?
cell culture cells that re-express telomerase onto the end of chromosomes which allows growth forever
Do immortalised cells follow the Hayflick limit ?
No
Do immortalised cells need growth factors or a solid support?
yes , both
What type of cell is a ‘TERTed’ skin fibroblast (used as mammalian cell culture)?
immortalised cell
What are transformed (mammalian) cells?
mammalian culture cells that have a loss of normal proliferation control
DO transformed cells need growth facotrs?
no (growth factor independent)
Do transformed cells die?
no - they are immortal
Do transformed cells undergo contact inhibition?
no
What is the impact of transformed cells not undergoing contact inhibition?
they can form multiple layers as opposed to monolayers like primary and immortalised cells
Where type of tissue are transformed cells derived from?
cancer tissue
HeLa, SHOS2, MCF7 and hybridomas for MAB production are all examples of what type of mammalian culture cell?
transformed cells
What two broad categories exist for getting DNA into eukaryotic cells?
physical
biological
Name the 4 physical methods of getting DNA into eukaryotic cells in the lab.
- electroporation
- transfection (chemical)
- microinjection
- biolistics
What name can be given to encompass most the different biological methods of getting DNA into eukaryotic cells? What is the one exception?
viruses
exception = bacteria (for GM plants)
What 4 categories of virus are used as biological methods of getting DNA into eukaryotic cells?
- DNA virus
- retrovirus (RNA)
- lentivirus (RNA)
- neurotropic virus (ssRNA)
Name two DNA viruses used for getting DNA into eukaryotic cells
- baculorvirus
- adenovirus
Give a common example of a retrovirus
HIV
Name two common neurotropic viruses used for getting DNA into eukaryotic cells
Zika virus
Rabies virus
What is the basis of electroporation for getting DNA into eukaryotic cells?
several 100V applied across a few mm
- causes transient permeabilisation of regions of the plasma membrane
What disadvantages are there to using electroporation for getting DNA into eukaryotic cells?
- expensive because i.e., need the kit
- high voltage frequently causes cell damage (therefore care is requires with the coditions
Describe the basis of CaPO4- transfection for getting DNA into eukaryotic cells.
Mix HEPES-buffered saline (HBS), CaCl2 and DNA
- produces CA2+ and PO42+ forming CaPO4 ppt
- DNA binds to the surface of the ppt
- taken up in pinocytic vesicles via micropinoytosis
What is the name of the cellular process that we take advantage of in order to get DNA into eukaryotic cells via CaPO4 transfection?
What is the basis of this process/what does it usually do?
micropinocytosis
- takes up liquids and macromolecules by engulfing them in pinocytic vesicles
What is an advantage of getting DNA into eukaryotic cells via CaPO4 transfection?
Cheap
(also simple and easy to carry out)
What is a disadvantage of getting DNA into eukaryotic cells via CaPO4 transfection?
low transfection efficiency
What is the basis of liposomal transfection?
Liposomes containing DNA fuse with the plasma membrane and rekease the DNA into the cytoplasm
What is the efficiency like of liposomes coating DNA?
high efficiency
e..g 90% effeciency transfection of COS7 cells
n.b. still variable dependent of the cell
What is the problem with normal liposomal transfection for getting DNA into eukaryotic cells?
high toxicity
because DNA is not meant to be in the cytoplasm
therefore triggers an innate immune response
What can be used be used to help liposomal transfection into primary cells and other cells that are often diffcult to transfect? How do they help this process?
Polyamines e.g. lipofectamine
the cationic polymer and plasmid compelx can be taken up via endocytosis
- then be degraded in the cytoplasm
(as opposed to fusion and release)
What conditions change for different transfections?
- cell type
e.g. how healthy the cell is or amount of DNA - cell density
- trnasfection reagent
- vector and insert combination
Why must transfection conditions be determined empirically each time transfection takes place?
There are lots of variables that change each time a transfection takes place
What is transient transfection?
short term ~1-7 days
How is stable/permanent transfection achieved?
long term selection using markers
ensures integration of vector and GOI inot the genome
(because integration is random)
describe the basic process of microinjection
- a pipette with suction holds a fertilised mouse egg
- microinjection of the vector directly into the nucleus
- forms transgenic mice
What is another name given to ‘Bolistics’ for getting DNA into eukaryotic cells?
“gene gun”
What are microprojectiles?
tiny metal particles (gold usually?) that are DNA-coated
Microprojectiles are suspended as a drop on what?
macroprojectiles
How are macroprojectiles impelled (fly) in bolistics?
- explosion with gunpowder
- or breakage of a membrane
- in a pressurised chamber (HELIOS gene gun)
In bolistics, what do macroprojectiles hit and what happens because of this?
they hit a stopping plate
- microprojectiles fall onto the tissue below
Is microprjectile bombardment used for transient or stable transfection?
either, used for:
- transient gene expression assays
- and also to generate permanently transformed cell lines or organisms
Wat is the name of the only technique so far this is successful in putting genes into plastids and mitochondria?
Bolistics/gene gun
How is a bolistics “gene gun” used in genetic immunisation?
impell genes for antigens into an animal’s skin
(Powderject)
up to biological methods
