Chapter 6 - Genetic Modification Flashcards
What is needed in order to modift cells in culture?
Gene of interest
Cells
Transfection mechenism
Selection mechanism
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?
Proliferating synkaryons
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).