91335 Lecture Notes - Lecture 4: Rna Polymerase Iii, Endosome, Provirus
MB2 Lecture 4
16/4/18
Eukaryotic expression systems and transgenes
Genes expression in mammalian cells
Genes encoding proteins that requires post-translational modifications must use eukaryotic
cells
Transfection – introduce gene into host cell using plasmid vectors
Transduction- viral vectors
Transiently- not permanent transformation, just for limited time the gene is expressed
Choosing mammalian expression vector
- Species and type of host cells available
- Is transient expression of foreign protein possible or will it be necessary to isolate
cell lines that express permanently
- Size of gene
- Presence of controlling elements in transfected DNA
Elements of mammalian expression vector
- Prokaryotic origin of replication for propagation and production of vector in bacteria
- Eukaryotic origin of replication for propagation and maintenance in transfected cell
lines
- Transcriptional elements
- Selectable markers
Introduction of recombinant vectors into mammalian cells
1. Calcium phosphate
Precipitation, mix with DNA to get fine precipitate and overlay in culture, cells transfected
via endocytosis
Primary cells- ells of ody, diffiult to grow i ulture, do’t orally grow easily, tuour
cells are mostly used
Cell line- propagate indefinitely in culture
Physical methods too harsh on primary cells
2. Polybrene
Polycation, stable transfection of low-molecular weight DNAs into cell lines
3. Liposomes
Encapsulation of DNA in membrane- endocytosis, can use on primary cells but is not very
efficient
4. Direct microinjection into nuclei
Small scale, used to establish cell lines,
5. Electroporation
Pierces cell wall with electrical pulse, harsh on primary cells
6. Gene gun
Shoots small particles though cell wall, injection of GM vaccines into muscle of human
Most of these are not very efficient
find more resources at oneclass.com
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Growth curve
Trypsin EDTA used to removed cells from culture plate
Transfection done in log phase (actively dividing)
Cloning cells
Monolayers for mammalian cells
multiwall dishes, petri dishes
grow up and introduce cells via mechanism
cell with selective marker (resistance gene) show transformed cells with resistant genes
clone of cells should all have same characteristics
nonviral gene transfer
disadvantages
- Harmful to primary cells
advantages of techniques
- No co-transfers of unwanted viral genes
- Physical methods more readily lend to manufacturing conditions
- Liposome-mediated closest to clinical application but is not stable or long term
Viral vectors
Tumour viruses cause transformation via transfer of genetic material
- SV40
- Adenoassociated: know where gene is going
- Herpes: large inserts, not used so far
- Retroviruses: gene therapy, avian, murine and human origin including lentiviruses
- Adenoviruses
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Genes encoding proteins that requires post-translational modifications must use eukaryotic cells. Transfection introduce gene into host cell using plasmid vectors. Transiently- not permanent transformation, just for limited time the gene is expressed. Species and type of host cells available. Is transient expression of foreign protein possible or will it be necessary to isolate cell lines that express permanently. Presence of controlling elements in transfected dna. Prokaryotic origin of replication for propagation and production of vector in bacteria. Eukaryotic origin of replication for propagation and maintenance in transfected cell lines. Introduction of recombinant vectors into mammalian cells: calcium phosphate. Precipitation, mix with dna to get fine precipitate and overlay in culture, cells transfected via endocytosis. Primary cells- (cid:272)ells of (cid:271)ody, diffi(cid:272)ult to grow i(cid:374) (cid:272)ulture, do(cid:374)"t (cid:374)or(cid:373)ally grow easily, tu(cid:373)our cells are mostly used. Physical methods too harsh on primary cells: polybrene. Polycation, stable transfection of low-molecular weight dnas into cell lines: liposomes.
