Playing With The Genome 29/11/22 Flashcards
What is a genetically modified organism (GMO)?
An organism that genotype has been altered by humans to change phenotype by adding foreign genetic material.
What are examples of GMO?
-Bananas
-Strawberries
-Alfalfa
-Corn
-Animals
What are the ethical concerns with GMO?
-Are they safe to consume
-Missuse
-Accidental unintended consequences
-Who owns the technology
-GM babies
What is a transgenic organism?
An organism containing foreign DNA. For example, adding crop resistance genes from one crop to another crop.
How to you make a genetically modified organism?
-Selective breeding (for example, artificial selection of carrots)
-Recombinant DNA technology
What is recombinant DNA technology?
Taking DNA from one place (same organism or different one) and rejoining the DNA in a way that gives the desired effect.
What is molecular cloning?
-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)
What happened in the history of molecular cloning?
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.
How do you select or identify the modified bacterial cells?
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.
What is the history of GMOs?
-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
What was used before human insulin was produced?
-Animal (pig) insulin was used. This wasn’t always effective as its not human.
What are the good and bad sides of recombinant human growth hormone being produced?
-Can have clinical benefits to cure illness
-Can be abused to help people grow more muscles (athletes)
What modern tools are used to clone?
-PCR techniques
-Engineered plasmids vectors
-Modified restriction enzymes for cutting specific gene sequences
-Competent bacterial strains
Why would you make a knockout mouse?
-To mimic a genetic disorder to study it
-To study gene function
What is a phenotype?
The observable traits of an individual resulting from the interaction between their genotype and environment.
What separate processes are needed to make a KO mouse?
-In vitro fertilisation
-Stem cell biology
-Recombinant DNA
-Genetic engineering or gene editing
-Sequencing technology
What is the process of making a KO mouse?
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.
What is a stem cell?
An undifferentiated cell which can specialise into any type of cell.
What is homologous recombination?
Exchange of genetic code between identical/similar (homologous) sequence.
What can homologous recombination be used for?
-A natural repair mechanism
-Can be used to insert desired DNA sequences
What is a common problem for biomedical research?
Making the KO mice can be embryonic lethal as the deleted gene may have been important in embryonic development.
How are KO mouse made when the gene modification is lethal?
-Conditional knock outs
-Conditional mutants
-Reporter mice
How can you control the deletion of the gene?
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.
What is cre-lox system?
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).
What is the different between cre and the Loxp site?
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.
How do the different types of Cre-LoxP work?
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.
What is tamoxifen?
It’s a drug used in chemotherapy for treating breast cancer.
How is cre modified in the lab?
Cre activity is induced in some way. Either by a promotor or a drug.
How is cre drug driven?
You can make tamoxifen induced creER (ER = oestrogen receptor) as this means that cre will only be induced when tamoxifen is added.
What is an example of conditional KO mice?
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.
What is the problem with cell specific cre?
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.
What is temporal gene deletion?
Deletion in the presence of a drug.
What is spatial gene deletion?
Deletion only where and when a promoter is active.
Can you have both temporal and spatial deletion?
Yes. Combining promoter driven expression with drug inducible cre recombination allows control of gene deletion.
