Genome Manipulation 1 Flashcards
Give types of genome modification.
Modifying expression levels, expression pattern and modifying gene/protein sequence.
What is meant by temporally modifying the gene expression pattern?
The gene is only expressed at a specific time and you want to express it at a different stage of development.
What is meant by spatially modifying the gene expression pattern?
The gene is only expressed in specific tissues and you want it expressed elsewhere.
Describe chemical random mutagenesis using EMS.
EMS is put in the food of drosophila, causes DNA damage in sperm. Progeny can have random mutations on any gene in the genome. These progeny are then used to establish stock line, and after a few generations 25% of the progeny will be homozygous mutants (mendelian inheritance). Screen for homozygous mutants.
How does EMS act as a mutagen?
It causes C-G to A-T pair transition, causing aa substitution, or causes a small deletion, resulting in a frameshift.
In what case would you screen using heterozygous?
If studying a recessive lethal allele.
What additional step needs to be done if using C.elegans for random chemical mutagenesis?
Bleach must be added in order to synchronise the life cycles of the C.elegans so that phenotype can be studied across all the progeny.
Give some advantages of using drosophila.
Cheap. Quick- only takes 12 days to get progeny. Developmental life cycle can give details on human development.
Give some features of drosophila that can be studied at high throughput.
lethality, body size, flight ability - high throughput as easy to study
How is the throughput usually increased?
By using computers to record and analyse the data.
Why can drosophila be used for transposon based random mutagenesis?
The P element (transposable) exists naturally in drosophila, if put into lab drosophila it will be able to insert itself into the genome.
Describe the P element.
Consists of 4 exons that code for both transposase and a repressor via alternative splicing. Alternative splicing is sex specific. Preferred insertion site is in the 5’UTR before the start of another gene.
How is the P element used to randomly modify the genome via insertion of a gene?
Inject plasmids into drosophila embryos. Vector plasmid contains a recognition sequence, the target gene and a white eye marker gene. The Helper plasmid contains the active transposase gene (from P element). Some cells take up both plasmids and are modified. Gene of interest has been integrated into the host genome, resulting in transgenic drosophila.
What happens to the transposase following random insertion of the target gene?
Transposase is eventually lost through cell division- not integrated into genome and only a limited number of copies were injected.
What is an enhancer trap?
When a transposon is inserted between enhancer elements found upstream of a gene.
