BIOL114 - Lecture 10

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What is a transgenic animal?
An animals that has additional genes (transgenes) introduced
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How are transgenic mice created?
Fertilised egg prior to fusion of the pronuclei. Use microinjection to insert foreign DNA. Eggs transferred into foster mother. Analyse offspring for presence of transgene (using PCR), breed positive mice to generate homozygous mice.
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What are the uses of transgenic animals?
Reporter genes to follow gene expression. As models of disease. To gain altered (desirable characteristics), for pharming, insect control and for general fun.
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What is a chondro mouse?
It is a transgenic mouse expressing green fluorescent protein under control of the collagen II promoter (reporter gene). Green fluorescence where collagen iii is being made
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What is the spinocerebellar ataxia mouse model?
It is where a transgene encoding for abnormal ataxin protein (neurotoxic) was put into a mouse.
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Give an example of altered characteristics due to transgenes:
The insertion of Human Growth Hormones in a transgenic mouse made is much larger than a normal mouse.
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What is a pharm animal?
It is a gentically modified animal that is used to make proteins that have medicinal value.
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How can inserts be genetically engineered?
The Aefes aegypti was genetically engineered to be sterile. These flies are typically a vector for dengue fever. But creating the sterile male, they compete with fertile males and cause a population crash.
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How are transgenic animals created for entertainment/fun purposes.
Multicoloured GloFish - in America, some fish have been created that glow under UV light as they contain various fluorescent proteins.
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What is a knockout animal?
In these animals, a wild type gene is replaced with an inactive gene to create a gene knockout
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What is a transgenic animal?
It s when there is insertion of exogenous DNA into random non homologous site in mouse genome, frequency high so relatively easy to produce such mice.
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What is a knockout mice?
There is insertion of modified DNA at a specific homologous site in mouse genome, frequency very low so difficult to produce such mice. Frequency very low so difficult to produce such mice
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What are the steps in creating a knockout mouse?
Remove cells from inner cell mass of black mouse embryo, culture ES cells, replace coding region of gene with DNA with neomycin resistance gene, add thymidine kinase gene to plasmid containing knocked out gene, Add recombinant plasmid to ES cells.
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Once you have added the recombinant plasmid to the cultured ES cells, what do you do?
You select for ells that have taken up plasmid with neomyin. Select against cells that have a non specific integration with ganciclovir. Inject knockout ES cells into blastocyst of white mouse. Implant blastocyst in foster mother. Identify chimeric.
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What does t mean to be chimeric?
f
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How do we knock out a gene?
We use Neomycin resistance gene as a marker. Addition of neomycin kills cells without this gene. Use Thymidine kinase gene as a marker. Addition of a drug, ganciclovir kills cells with TK gene.
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What are the uses of knockout mice?
To test gene function, as models of human diseases e.g. CFTR, Dystrophin - Duchenne muscular dystrophy, globin - thalassaemia.
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What is gene therapy?
Treating diseases with gene. A mutant gene is replaced, or supplemented, by a normal one, or therapeutic gene is introduced (e.g. anti-cancer gene) in vivo or ex vivo approaches.
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What are the targets of gene therapy?
Single gene disorders, cancer, rheumatoid arthritis, erythropoietin for kidney patients
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What did the early attempts at gene therapy target?
Cystic Fibrosis - in vivo approach
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How was the early attempt at CF gene therapy conducted?
Wild type cystic fibrosis gene was added to adenovirus vectory, squirted aerosol containing virus into lungs of CF patients,
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Did the early gene therapy show improvement? Did it work?
Yes - for 8 weeks
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What happened after 8 weeks?
The host immune system attacked cells carrying virus and wiped them out - removing normal cystic fibrosis gene and any chance of cure.
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What are the problems associated with using the adenovirus as a vector?
The adenovirus is a common cold virus and we all have antibodies against it. Adenovirus may elicit severe immune response. Adenovirus inserts randomly into genome. Adenovirus only inserts into chromosomes if the host cells actively replicating DNA
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What is good about the adeno-associated virus?
It is very small (doesn't elicit much immune response), infects both non dividing and dividing cells. Vectors developed to control protein expression.
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What is SCID?
Severe combined immunodeficiency disease
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How is stem cell gene therapy conducted?
Insert RNA version of normal allele into retrovirus. Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Inject engineered cells into patient
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Using retrovirus as a vector, what was the risk for SCID patients?
Leukaemia
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What has been done to remove the possibility of cancer?
Safer vectors are now in use
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What does gene editing involve?
Making a very small specific change in exactly the gene that you are interested in. This is done using the CRISPR cas 9 system.
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How many components does the CRISPR-Cas9 system have and what are they?
2 - CRISPR component (RNA molecule that acts as a guide, it will base pair with points of interest), CAS9 enzyme (a nuclease) cuts double stranded DNA. The position it cuts DNA is determined by CRISPR
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A break is made by the enzmye, what then happens?
The break is repaired with crossing over of the normal DNA. This introduces a normal version of the gene into the chromosome.
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There is another way of repairing the break, what is this?
Non-homologous end joining. Much more error prone. There is no template and the ends are joined together. You can have lost gene, or an insertion at the point of the break. This is useful in gene therapy if you have a disease caused by an abnormal fo
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How can you silence a gene?
You can silence it by mutating it further.
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How are transgenic mice created?

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Fertilised egg prior to fusion of the pronuclei. Use microinjection to insert foreign DNA. Eggs transferred into foster mother. Analyse offspring for presence of transgene (using PCR), breed positive mice to generate homozygous mice.

Card 3

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What are the uses of transgenic animals?

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Card 4

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What is a chondro mouse?

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Card 5

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What is the spinocerebellar ataxia mouse model?

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