L9 Introduction and maintenance of exogenous DNA into host cells Flashcards
Saccharomyces cerevisiae
single cells
yeast
Aspergillus nidulans
multinucleate hyphae
fungi
Drosophila melanogaster
P-elements
Arabidopsis thaliana
Ti Plasmid
Mus musculus
Embryonic stem cells
Yeast transformation
- uninucleate cells divide by budding
- cell wall degrading enzymes
- sphaeroplasts - osmotically sensitive
- DNA + Ca2+ + PEG => transformations (creates small pores in cell membrane)
YIp
See OneNote diagram
Bacterial plasmid backbone + yeast selectable marker
- YIp integrates into the genome by homology
YEp/YRp
YIp + yeast origin of replication
- high transformation freq.
- not integrated
- high copy number (~30 per cell) but poor segregation during cell division as it does not have a centromere
- maintained and replicates independently, recombination not required
- shuttle vector
YEP - high copy number, natural yeast plasmid
YRp - yeast chromosome, low copy number BUT the SAME with regards to TRANSFORMATION
YCp
YEp/YRp + centromere
- high transformation freq.
- good segregation
- single copy/cell
YAC
yeast artificial chromosome
- can be used to clone very large DNA fragments
- stability increases and copy number decreases with size
Yeast selectable markers
See OneNote diagram
- mainly WT amino acid auxotrophic markers
Yeast transformants
- Transformants are due to HOMOLOGOUS recombination either by single or double cross overs
single => left with both WT, mutant and part of the plasmid, addition transformants
double => WT replaces mutant copy, less common as it requires double recombination, substitution transformants
Plasmids with yeast origin of replication
maintained in the nucleus but are replicated independently of the yeast genome, integration is limiting for YIp vectors
Using transformation to clone a gene by complementation
See OneNote
- figure out what the mutant is
Genomic library
See OneNote
Collection of shuttle vectors that contain different sections of that genome => shuttle vector then inserted into the yeast mutant
Can isolate the shuttle vectors as they are replicating independently and put it back into E.coli in order to make more copies that we are able to sequence and find out the exact sequence that complemented our mutant - subclone to define minimal complementing region
Do you need to know if your mutation is dominant or recessive?
Yes
Using transformation to investigate gene expression with a reporter
See OneNote
- vary deletion region and assay for expression
- Region of interest can be transferred into a vector and analysed
Using transformation for overexpression/gene dosage experiments
- use different plasmids to vary copy number of your gene
- Different vectors have different copy numbers so would produce different amount of product
OR
- use highly expressed promoter to drive expression of your gene
Using transformation to characterise gene function in yeast
gene inactivation and gene replacement - specific mutations by homologous recombination
Gene inactivation - strategy 1
See OneNote
- insert selectable marker into the gene to be inactivated on a YIp plasmid => gene cut in half => inactivated
- double X over
Gene inactivation - strategy 2
See OneNote
- insert internal region only of gene into YIp vector
Gene inactivation constructs
See OneNote
What if the inactivation is lethal?
Use a diploid recipient e.g. Aspergillus => heterozygous gene knock-out
Shift to poor nutritional conditions to induce sporulation
Genotypic ratio of heterozygous knock out
2 ura3- : 2URA3+ = not essential
2 ura3- : 0URA3+ = eseential
Gene inactivation
to create loss of function mutant
Gene replacement
See OneNote for steps
to introduce more subtle mutations into the genome
A genetic way of ensuring integration at the gene of interest?
- use Aspergillus transformation
Aspergillus transformation
See OneNote
- Rapid, generates protoplasts, add DNA, isolate transformants
- Auxotrophic and drug resistant selectable markers
Aspergillus nidulans
- Ascomycete
- Haploid or diploid
- Asexual and sexual cycles BUT multinucleate hyphae
A.nidulans integration
non-homologous integration occurs a lot more often than homologous integration (unlike in yeast)
Non-homologous»_space; Homologous ad. and disad.
Advantages
- can introduce any gene (no homology)
- can leave native gene intact
- can introduce multiple copies of gene
Disadvantages
- possible positions effects on expression
- homologous events more difficult to isolate
Gene targeting in Aspergillus
See OneNote
- targeting DNA to the argB locus
Aspergillus conidiation
- asexual life cycle
- ordered developmental program
- requires the product of the brlA gene
- brlA mRNA produces only with the onset of conidiation (encodes a TF)
Does brlA promote conidiation or does conidiation promote brlA?
See OneNote
Aspergillus transformation
- high freq. transformation => clone by complementation
- DNA must be integrated, most integrations are non-homologous
- homologous integrations do occurs => gene targeting and inactivation are possible
- reporter genes can be easily introduced
Ku70/Ku80
- required for DNA repair by NHEJ
- identified and inactivated A.nidulans nkuA (A.nidulans homologue of the human Ku70)
- in inactivated nl=kuA, integrations were essentially 100% homologous => gene inactivation/replacement now east
