Playing With The Genome 29/11/22

Question 1

What is a genetically modified organism (GMO)?

Answer 1

An organism that genotype has been altered by humans to change phenotype by adding foreign genetic material.

Question 2

What are examples of GMO?

Answer 2

-Bananas
-Strawberries
-Alfalfa
-Corn
-Animals

Question 3

What are the ethical concerns with GMO?

Answer 3

-Are they safe to consume
-Missuse
-Accidental unintended consequences
-Who owns the technology
-GM babies

Question 4

What is a transgenic organism?

Answer 4

An organism containing foreign DNA. For example, adding crop resistance genes from one crop to another crop.

Question 5

How to you make a genetically modified organism?

Answer 5

-Selective breeding (for example, artificial selection of carrots)
-Recombinant DNA technology

Question 6

What is recombinant DNA technology?

Answer 6

Taking DNA from one place (same organism or different one) and rejoining the DNA in a way that gives the desired effect.

Question 7

What is molecular cloning?

Answer 7

-Need a way to copy your sequence of interest
-Take section of DNA using restriction enzymes (for example, gene for insulin)
-Clone it into a bacterial chromosome (plasmid) so you have recombinant DNA
-Transgenic bacterium with plasmid containing insulin gene
-Transform bacteria to express your cloned sequence (for example, secreting human insulin)

Question 8

What happened in the history of molecular cloning?

Answer 8

Discovery of different tools such as DNA ligase (joins DNA together) and restriction enzymes (cut the DNA in specific places). Development of processes such as PCR and USER cloning.

Question 9

How do you select or identify the modified bacterial cells?

Answer 9

Add a antibiotic resistant gene to the recombinant gene plasmids and then at the end add antibiotics and none modified plasmids will die and you’ll be left with only transformed bacteria.

Question 10

What is the history of GMOs?

Answer 10

-1987 first KO mouse
-1994 first conditional KO mouse
-2013 first CRISPR to edit germline in a mouse
-2018 germline editing of humans with CRISPR

Question 11

What was used before human insulin was produced?

Answer 11

-Animal (pig) insulin was used. This wasn’t always effective as its not human.

Question 12

What are the good and bad sides of recombinant human growth hormone being produced?

Answer 12

-Can have clinical benefits to cure illness
-Can be abused to help people grow more muscles (athletes)

Question 13

What modern tools are used to clone?

Answer 13

-PCR techniques
-Engineered plasmids vectors
-Modified restriction enzymes for cutting specific gene sequences
-Competent bacterial strains

Question 14

Why would you make a knockout mouse?

Answer 14

-To mimic a genetic disorder to study it
-To study gene function

Question 15

What is a phenotype?

Answer 15

The observable traits of an individual resulting from the interaction between their genotype and environment.

Question 16

What separate processes are needed to make a KO mouse?

Answer 16

-In vitro fertilisation
-Stem cell biology
-Recombinant DNA
-Genetic engineering or gene editing
-Sequencing technology

Question 17

What is the process of making a KO mouse?

Answer 17

1) Embryonic stem cells are cultured from a mouse pre-implantation embryo.
2) Construct a vector. Homologous DNA will be on either side of the foreign DNA wanting to be inserted.
3) Stem cell transfection. This will contain pieces of DNA that are homologous to native DNA so that DNA machinery recognises DNA and adds it to the gene. The foreign DNA will be in between the homologous DNA so it will be inserted into the gene and the original gene will be knocked out.
4) Proliferation of targeted ES cells as selection for presence of the gene is looked for.
5) The ES cells are injected into blastocyst and placed into the female mouse to be born. This creates a mosaic mouse pup.
6) Mosiac mouse are bred until you have a fully KO mouse.

Question 18

What is a stem cell?

Answer 18

An undifferentiated cell which can specialise into any type of cell.

Question 19

What is homologous recombination?

Answer 19

Exchange of genetic code between identical/similar (homologous) sequence.

Question 20

What can homologous recombination be used for?

Answer 20

-A natural repair mechanism
-Can be used to insert desired DNA sequences

Question 21

What is a common problem for biomedical research?

Answer 21

Making the KO mice can be embryonic lethal as the deleted gene may have been important in embryonic development.

Question 22

How are KO mouse made when the gene modification is lethal?

Answer 22

-Conditional knock outs
-Conditional mutants
-Reporter mice

Question 23

How can you control the deletion of the gene?

Answer 23

You can control what cells the deletion occurs in or when the deletion of the gene occurs. This is done using the cre-lox system.

Question 24

What is cre-lox system?

Answer 24

Cre-Lox recombination is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is found in nature and is a bacteriophage.
It’s made of cre-recombinase and the Loxp Site (Flox).

Question 25

What is the different between cre and the Loxp site?

Answer 25

Cre-recombinase are the molecular scissors and Loxp is a 34 base pair sequence that instructs where the cut by recognising the DNA tags placed by cre-recombinase.

Question 26

How do the different types of Cre-LoxP work?

Answer 26

The protein Cre-recombinase recognizes 34 bp loxP sites, and the orientation and location of the loxP sites determines how the genetic material will be rearranged.

Inversion: If the loxP sites are on the same DNA strand and are in opposite orientations, recombination results in an inversion and the region of DNA between the loxP sites is reversed.
Deletion: If the sites face in the same direction, the sequence between the loxP sites is excised as a circular piece of DNA (and is not maintained).
Translocation: If the sites are on separate DNA molecules, a translocation event is generated at the loxP sites.

Question 27

What is tamoxifen?

Answer 27

It’s a drug used in chemotherapy for treating breast cancer.

Question 28

How is cre modified in the lab?

Answer 28

Cre activity is induced in some way. Either by a promotor or a drug.

Question 29

How is cre drug driven?

Answer 29

You can make tamoxifen induced creER (ER = oestrogen receptor) as this means that cre will only be induced when tamoxifen is added.

Question 30

What is an example of conditional KO mice?

Answer 30

SOX9 is believed to be a core regulator of ECM genes during fibrosis. However, if this gene is knocked out before the mice develops then the mouse won’t develop properly. Therefore, conditional KO is used to KO SOX9 once the mouse is an adult and the effect on fibrosis can be investigated.

Question 31

What is the problem with cell specific cre?

Answer 31

Can’t grantee that only specific cells are affected so you may kill off other cells during development. You can’t be 100% certain that the promoter isn’t active in other cell types.

Question 32

What is temporal gene deletion?

Answer 32

Deletion in the presence of a drug.

Question 33

What is spatial gene deletion?

Answer 33

Deletion only where and when a promoter is active.

Question 34

Can you have both temporal and spatial deletion?

Answer 34

Yes. Combining promoter driven expression with drug inducible cre recombination allows control of gene deletion.