L24 - gene cloning in eukaryotes 2 Flashcards
(24 cards)
what are the applications of expressing heterologous genes
produce useful proteins in large quantities bypassing tedious purificationn from low-yielfing sources
= also allows proper post-translational modifications
define transfection
proscess which DNA is taken up and expressed by eukaryotic cells
= ‘transformation’ is decribing the cell chnaging phenotype NOt taking up new genetic material
define selection
proscess by which transfected cells are isolated from non0transfected cells
= which have taken up our desired DNA
give a few considerations when designing DNA-mediated gene transfer
Vectors:
- BACs or YACs, or viral vector (short term)
- choose most apropraite method based on size or time
Transfection method:
Selectable marker:
- allows us to tell if DNA has been sucessfully taken up
Stable vs Transient transfection:
long or short term transfer of DNA
Regulated promoter:
- do we need/want to control gene xpression/toxic prouct for example
name a common shuttle vector used in eukaryotic gene cloning
pcDNA
give the chareceristics of pcDNA shuttle vector and split between useful for eukaryotic and prokaryotic cloning or both
plasmid vector
Both:
- MCS - multiple digest regions that can be selected based on insert DNA/gene
- T7 promoter - used to check whether insert has been correcly inserted and is functional - +/- selection = derived from bacteriophage
Eukayrotic:
- Cytomegalovirus/promoter for MCS allowing gene expression for insert
- Poly A signal sequence - stops translation AND adds the poly A tail
- Neomycin/Antibiotic resistance gene with promoter/SV40 - for selction in mammilian cells
Prokaryotes:
- pUC orogin of replication in E.coli - good for creating multiple copies of gene
- Antbiotic resitsance gene/ampicillin for selction in E.coli
describe a property of the T7 promoter in pcDNA shutle vector
within the MCS
= the T7 promoter can either be sense or anti-sense
= it can run either direction based on needs/the inert
what is the kozak consensus sequence and how can it help in expression plasmids
massively improves translational efficiency of cDNA
= sequnce more strongly recognised by ribosome
= more of desired protein is produced from vector
describe Neomycin phosphotransferase as a selectible marker
enzyme phosphoylates neomycin/antibiotics inactivating them
= ANTIBIOTIC RESISTANCE GENE
= used for positive/negative selection
describe the Herpes simplex virus thmidine kinase (HSV-TK) selectible marker
this gene phosphorylates a normally non-toxic compound
= when phosphorylted prevents proliferation of cell
= if gene is present and active = cell dies
describe chemical DNA-mediated transfection
DNA mxied with chemicas to form compleexes
= allows bidning to cell surface causing endocytosis
describe lipofection as a form of transfection
DNA mixed with lipid to form artificial liposome = small vesicles
= more efficientyly endocytosed than DNA-mediated
describe a physical method to transfect DNA into a cell
microinject DNA directly into nucleus
= most efficient method BUT may cause physical stress to the cell/damage
= 1 cell at a time
describe electroportation as way of transfection
apply brief electric shock to cell to form transient/short-lived pores
bathe cell in solution with DNA
= DNA enters pores
describe the method of transfection with viral infection
infect the cell with DNA iuntegrating it into genome OR just short lived
= adenoviruses = transient
describe the regulated gene expression/background info of the Tet gene
Tet provides antibiotic resistance to Tetracycline
= when not in contact with antibiotic the Tetanus gene is repressed/switched off
= repressor gene produces protein that binds to gene ooperator
what gene we use to have ‘regulated expression vectors’
Tet gene and Tet Repressor on seperate plasmid
= 2 plasmids = 1 with Tet + 1 with Tet(r)
describe the Tet-on system for a controlled gene expression
2 plasmids within the same cell each witha part of the tet system
= 1 with tet gene with the gene of interest after it + 1 with repressor
tetracycline added –> repressor is removed from tetracycline gene operator –> polymerase can bind and transcribe THE GENE OF INTEREST
= due to it being after the TetO
2 types of viruses that can be used as vectors
adenovirus –> transient
Lentivirus –> stable
= viruses are naturally good at transferring genes = do at a high efficiency
describe an adenovirus and the different generations and what they remove
contains linear dsDNA genome
- made of a number of regions that are transcribed in sequence due to being linear
certain elemnets can be removed to improve safety and transfection
1st gen vector:
could replicate by itself but removed E3 regions for safety
2nd gen:
coulkd NOT replicate without presecence of another virys with the removed E2 genes = safer
3rd gen:
nearly all viral genes removed = need helper virys and cell line that supplies proyeins for replication
describe methgod to insert gene/DNA into a adenovirus for transfection
- digest plasmid and insert and ligate into MCS
- tranform/insert into E.coli
- transfect into viral cell/adenovirus
= adenoviruses do NOt insert/integrate into the host cells genome
= transient/short lived production of protein
describe the Lentiviral (retroviral) life cycle
- viral surface glycoproteins bind to cell membrane and insert viral RNA genome
- ssRNA converted into dsDNA by viral reverse transcriptase
- viral DNA eneters nucleus and integrase inserts into host geneome
= viral proteins are produced and can infect other cells
= stable/long term/permanent infection
what must we do when using lentivirus/retrovirus transfection
the virus is split into 3 seperate plasmids for safety regions
- packaging plasmid = Gag
- envelope plasmid = Env
- Transfer plasmid = Pol
all 3 seperate plasmids inserted into the virus
describe the method/work flow of creating Lentiviral vectors
- plasmid vectors are inserted into host cell
- viral proteins produced from the palsmids using the host/packaging cells machinery
- new viral particles produced that are removed and can be used to infect cells of choice
= these viruses will infect the new cells with gene of interest
