mammalian transfection

Question 1

what is transfection?

Answer 1

putting DNA or RNA into a mammalian cell

Question 2

true or false, mammalian cells dont take up DNA very readily, so they’re very delicate

Answer 2

true

Question 3

what are some challenges to introducing DNA into mammalian cells?

Answer 3

-nucleus acts like a double protector, not only does the cell have to cross cell membrane, it also has to get into the nucleus (barrier)
-mammalian cells destroy foreign DNA very readily
-A lot more DNA is required, which makes the process much more expensive (bacteria only needs a small amount of DNA, mammalian needs a lot)

Question 4

does mammalian transfection require a mini prep or a maxi prep?

Answer 4

maxi prep cause we need much more purified DNA

Question 5

what is the chemical transfection method?

Answer 5

we coat dna with positive ions, which form a complex, that can then fuse with the plasma membrane of the cell and enter by endocytosis. when it enters, it is in an endosome, and must escape it, in order to enter the nucleus and get transcribed

Question 6

what do we mean when we say that transfection is often transient?

Answer 6

-its often temporary. cells will not keep the DNA. we have a certain window where we can do experiments before its degraded

Question 7

why do endoscopes destroy DNA?

Answer 7

because they fuse with lysosome, who have hydrolytic enzymes that destroy DNA

Question 8

why does chemical transfection require a lot of DNA?

Answer 8

because a lot of it gets degraded because of the endosome

Question 9

what are the three types of chemicals that we can use for transfection?

Answer 9

-cationic liposomes
-calcium phosphates
-cationic polymers

they attach to dna and form a complex!

Question 10

which chemical is the best and which is the worse for transfection?

Answer 10

-best: cationic liposomes. they’re expensive but very efficient
-worst: calcium phosphates: very cheap but inefficient

Question 11

what is biological transfection?

Answer 11

we make viruses with recombinant DNA that can go infect a cell and give that cell that DNA

Question 12

what do you need for biological transfection?

Answer 12

-3-4 plasmids
-a packaging cell
-transfection reagent
-cell you’re trying to transfect

Question 13

in biological transfection, what genes are contained in your plasmid?

Answer 13

-gene of interest that you want to transfect
-packaging machinery proteins
-necessary viral proteins
-they contain the genes necessary to produce a virus that can infect another cell but cannot replicate (viral replication in the cell could be dangerous to other cells)

Question 14

do we need 3-4 plasmids in biological transfection?

Answer 14

instead of putting all viral proteins in one plasmid they put in 3 so that virus cant self assemble

Question 15

what are the steps to biological transfection?

Answer 15

1) packaging cells are transfected with 3-4 plasmids encoding the gene of interest, packaging machinery proteins and necessary viral proteins

2)virus is assembled in the packaging cells containing gene of interest. it is then harvested then purified

3) virus infects the host cells, releasing the gene of interest

4) protein expression

Question 16

how can biological transfection introduce the gene of interest into the genome of the mammalian cell?

Answer 16

-we can use retroviruses, as they create a provirus by inserting DNA into the host cell genome