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ANAT3231: lectures overview
Dr Annemiek Beverdam – School of Medical Sciences, UNSW Wallace Wurth Building Room 234 – [email protected]
“mature, differentiated cells can be reprogrammed to become pluripotent”
Oct4 Sox2 c-Myc Klf4
Stimulus-Triggered Acquisition of Pluripotency (STAP) (Obokato 2014, controversial)
Stem Cell Sources Embryonic vs Adult Stem Cells
iPS Cells
- Can generate any cell type - Easy to generate, maintain
and grow in lab - Perfect genetic match to
patient
- May retain age of parental cell
- Inheritance of mutations: teratomas
- May retain age of parental cell
- Inheritance of mutations: teratomas
The Future of Regenerative Medicine
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Future Stem Cell Technologies
1- how we can induce and maintain pluripotency? 2- how we can direct differentiation? 3- how we can cure diseased cells? 4- how we can repair mutations in cells?
Future Stem Cell Technologies
How can we direct differentiation? - Uncontrolled differentiation
- Directed differentiation
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Future Stem Cell Technologies Directed differentiation of cardiomyocytes
Mummery et al., Circ Res 2012
Future Stem Cell Technologies Directed differentiation of motor neurons
Dong et al., Nature 2014
Pluripotent stem cells
Future Stem Cell Technologies Directed differentiation of pluripotent stem cells
Future Stem Cell Technologies
How can we direct differentiation?
Directed differentiation: learn from developmental biology!
Future Stem Cell Technologies
How can we cure disease?
Disease Modeling and Drug discovery
(personalized medicine)
Future Stem Cell Technologies
How can we repair mutations in cells?
Gene Therapy:
Knock out technology
CRISPR/CAS9 genome editing
Crossing over is a natural process that happens during meiosis
Knock out technology = directed homologous recombination in pluripotent ES cells
Knock out technology
Endogenous gene
ATG
neoR
= regions of homologous DNA sequence
neoR
Targeting vector
Knock out allele
Homologous recombination
Knock out technology
Knock out technology
1 Creating knockout DNA construct
3. Heterozygous mutant ES cells
2. Generating embryonic stem cells
Chimeric mice
Genetic crosses to obtain Homozygous mutant mice
Heterozygous mutant Embryonic stem cells
Knock out technology
Knock out technology Engineering of targeting vectors
Repair mutations
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Knock out technology Engineering of targeting vectors
Expressing multiple genes from same GM locus
Gene X
Gene X
Knock out technology
Allows us to:
Study gene function in mice
Repair mutations or to express a second protein from a GM locus in ES cells
However:
Heterozygosity in recombined ES cells
(Low rates of homologous recombination)
CRISPR/Cas9 Genome Engineering (Clustered Regularly Interspaced Short Palindromic Repeats)
Guide RNA and Cas9
http://www.youtube.com/watch?v=0dRT7slyGhs
CRISPR/Cas9 Genome engineering Repair
Homology-directed repair: Provide donor template with homology arms