Chapter 6 - Genetic Modification

Question 1

What is needed in order to modift cells in culture?

Answer 1

Gene of interest

Cells

Transfection mechenism

Selection mechanism

Question 2

What is a dominant marker and when is it used?

Answer 2

These are markers that the cells don’t already have and and used for wild type cells

Question 3

What is a selectable marker and when is it used?

Answer 3

Selectable marker codes for an enzyme that reverts a mutant back to wild-type. Used for mutant cells as a selection mechanism.

Question 4

What is cell fusion?

Answer 4

Getting DNA into cells at the genomic level

Question 5

How do we get cells to fuse?

Answer 5

By using agents that promote cell fusion called fusogens. There are 3 main types of fusogens: biological, chemical and electrofusion.

Question 6

How do fusogens work?

Answer 6

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.

Question 7

What is a homokaryon?

Answer 7

A multinucleate cell in which all neuclei are the same.

Question 8

What is a heterokaryon?

Answer 8

A multinucleate cell with two or more different nuclei

Question 9

What is a synkaryon?

Answer 9

A mononucleate cell that has formed from a heterokaryon or a homokaryon

Question 10

What is a cell hybrid?

Answer 10

Proliferating synkaryons

Question 11

What is isolated chromosomes?

Answer 11

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

Question 12

What are microcells?

Answer 12

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.

Question 13

What is transfection?

Answer 13

The transfer of nuclein acids into an animal cell

Question 14

How is DNA intorudced into cells through microinjection?

Answer 14

Inject a known amount of DNA into cells via a micro-needle into the nucleas of a individual cell

Question 15

What sorts of artificial (indirect) methods are there to introduce DNA into cells?

Answer 15

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).

Question 16

What are some viruses used as a natrual method of introducing genes into a cell?

Answer 16

SV40

• 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

Question 17

What are some examples of genetic variant cells?

Answer 17

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.

Question 18

How does HAT media select for cells with transfected DNA?

Answer 18

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.

Question 19

How are transfected WT cells selected?

Answer 19

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.

Question 20

What types of expressions are there?

Answer 20

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)