What is needed in order to modift cells in culture?
Gene of interest
What is a dominant marker and when is it used?
These are markers that the cells don't already have and and used for wild type cells
What is a selectable marker and when is it used?
Selectable marker codes for an enzyme that reverts a mutant back to wild-type. Used for mutant cells as a selection mechanism.
What is cell fusion?
Getting DNA into cells at the genomic level
How do we get cells to fuse?
By using agents that promote cell fusion called fusogens. There are 3 main types of fusogens: biological, chemical and electrofusion.
How do fusogens work?
They cause proteins in the membrane to move around creating a region of the cell membrane with no proteins. These areas are unstable areas that allow for the cell membranes to fuse.
What is a homokaryon?
A multinucleate cell in which all neuclei are the same.
What is a heterokaryon?
A multinucleate cell with two or more different nuclei
What is a synkaryon?
A mononucleate cell that has formed from a heterokaryon or a homokaryon
What is a cell hybrid?
What is isolated chromosomes?
A method of chromosome transfer that involves isolating the at metaphase with colchicine for direct transfer.
Not very effective since the chromosomes get broken up easily by the proteolytic enzymes in FBS
What are microcells?
Microcells is a method of transfering chromosomes to another cell. Mitosis is arrested and results in the formation of small nuclei due to instability. The small nuclei are then treated with cytochalasin B which interrupts microfilaments and teh cytoskeleton. The conconction is then centirfuged and fusogens are used to introduce microcell into another cell.
What is transfection?
The transfer of nuclein acids into an animal cell
How is DNA intorudced into cells through microinjection?
Inject a known amount of DNA into cells via a micro-needle into the nucleas of a individual cell
What sorts of artificial (indirect) methods are there to introduce DNA into cells?
Calcium phosphate method - Co-precipitation of DNA with calcium phosphate will produce a very fine precipitate which the cell takes up
Diethylaminoethyl dextran - DNA and DEAE-dextran, treat cells with DMSO and cells take up DNA.
Liposomes - Formed using a positively charged lipid and DNA. This is readily taken up by cells.
Protoplast fusion - GOI is inserted into plasmid that is grown in bacteria (ie. E.coli). Remove cell wall with lysozyme and fuse protoplast with animal cell using polyethylene glycol.
Electroporation - Expose cells to high levels of electrical impulses which leads to reversible increase in membrane permeability.
Microprojectiles - Coat projectiles with DNA and shoot into cell (mainly used for plant cell transfetion).
What are some viruses used as a natrual method of introducing genes into a cell?
- simian virus (naturally infects monkeys)
- many known cleavage sites by RE
- 2 phases of transcription: early and late phases
- host cell needs to be derived from a monkey
Retroviruses (RNA viruses)
- can incorproate genes into nuclear DNA
-Formed complementary DNA (cDNA) using reverse transcriptase
What are some examples of genetic variant cells?
Lesh-Nyhan syndrome - cells that lack the enzyme Hypoxanthine-guanine phosphoribosyl transferase. These HGPRT- cells must rely on the de novo pathway in order to synthesise nucleotides and survive.
8-azaguanine and 6-thioguanine - analogoues of guanine and lethal when incorporated into DNA with HGPRT. Cells that grow in presence of guanine analoguous are HGPRT- mutants.
Bromodeoxyuridine (BUdR) Resistance - BUdR is an analogoue of thymidine which can be incorporated into DNA with Thymidine Kinase. Cells that can grown in BUdR are TK- mutants.
How does HAT media select for cells with transfected DNA?
HAT media is comprised of hypoxanthine, aminopterin and thymadine. When the gene of interest and marker (HGPRT or TK) gene is transfected in the cell, only then are they able to grown on the HAT media. All of the cells without the GOI interest and marker will not grown in HAT media.
How are transfected WT cells selected?
Dihydrofolate redeuctase (DHFR) - cells with DHFR genes exhibits increased resistance to methotrexate which blocks the de novo pathway of purine synthesis. Introduce to cells using calcium - phosphate coprecipitation method. The transfected cells are grown on medium high in methotrexate. Those with DHFR genes survive.
NeoR gene - codes for amino-glycoside phosphotransferawse and provides resistance to aminoglycoside antibiotics (ie. G418). NeoR gene is introduced via calcium-phosphate co-precipitate method.
Xanthine-guanine phosphoribosyltransferase (XGPRT) - a bacterial enzyme that converts xanthine to xanthine monophosphate. Plate cells on medium with hypoxanthine, xanthine, aminopterin and mycophenolic acid. Only cells with XGPRT will grow.
What types of expressions are there?
Transient expression - genes are expressed for a short period of time after transfection
Permanent expression - stable expression of a transfected gene (covalently ligate non selectable gene to selectable one & cotransfection)