How to introduce DNA to animal cells? - (Oct/12/2007 )
This question is a part of my labreport and I cant seem to find any good answers in my book.
Question: "There are a number of ways to introduce DNA to animal cells. Describe shortly 3 techniques and discuss advantages and disadvantages. What is the major problem of expressing a eukaryotic protein e.g. from an animal in a prokaryotic system?"
I have found some techniques:
1. Calcium phosphate precipitation
2. Direct micro injection
3. Retrovirus infection
5. Particle gun delivery
But wich ones are the most commonly used and what are the advantages/disadvantages?
Thanks for the help.
Lipofection is quite commonly used. Electroporation is nice and not so unusual but you need the apparatus, which can be expensive (or cheap if you cobble it together). Direct microinjeciton works very well for embryos microinjected at the single-or-few cell stage, but is VERY tedious in cell cultures. Calcium phosphate works but is an older technique, less commonly used now. Particle gun is quite uncommon but in use. Viral delivery takes a bunch of work but can preferentially deliver into particular cell types (but you need to find a virus that is amenable to manipulation which targets your cells of interest).
As for the second part of the question, consider the process of protein maturation in a eukaryote vs. a prokaryote. In a eukaryote, what happens to many proteins after translation?
Thanks. I've been focusing on lipofection and electroporation, but still can make my mind up about the third one. I was thinking, is heat chock proteins commonly used? And what about gene gun? Does anybody do transfection by calcium phosphate now a days?
"As for the second part of the question, consider the process of protein maturation in a eukaryote vs. a prokaryote. In a eukaryote, what happens to many proteins after translation?"
I still can't find the answer on this question. All I know is that the transcription and translation are coupled in a prokaryotic cell, since there are no nucleus. Coupled = The translation begins while the mRNA is still being synthesized. And in a eukaryotic cell, transcription and translation are spatially and temporally separated. Transcription occurs in the nucleus, and translation occurs in the cytoplasm. After translation in a eukaryotic cell the proteins are often folded to assume native secondary and tertiary structures. It the proteins aren't folded right they will be labelled with ubiquitin will eventually be destroyed.
The third one? You can chose retroviral or other transfection technique with selection process to get stable expression cell lines. Problem will be optimize codon usage, adding promoter and terminater for eukaryotic expression. You need another set of expresion unit for antiboitic gene for selecting stable line.