Lecture 2 Flashcards
what are the 2 major technologies we could use to manipulate stem cells?
1) genetic technologies
2) reproductive technologies
what 3 methods fall under genetic technologies?
1) artificial chromosomes
2) genetic manipulation (CRISPR etc)
3) sequencing
what 5 methods fall under reproductive technologies?
1) IVF
2) ES cells
3) iPS cells
4) in vitro gamteogenesis
5) cloning
what is a problem with mishandling embryonic stem cells?
they can lose their ability to transfer into the germ line
what is a knock out?
where a gene of interest is knocked out of the operon (no longer expressed) by terminating the gene within the stem cells
- can be replaced with another gene creating a tissue specific reporter
what are knock ins?
a new gene is fused into the position of the genes of interest
- you can signal where the sequence needs to be cleaved for specific genes so only part of the transcript is transcribed producing only the protein of interest
what is a pronuclear microinjection?
where multiple random copies of a gene are inserted into the nucleus of a cell and then planted into the oviduct of a pseudo pregnant female - not generally used anymore
what are the 4 parts of a human artificial chromosome?
1) telomeres
2) centromere
3) replication origin
4) gene
what are the 3 differences between a transgenic pronuclear microinjection and a knockout targeted gene insertion?
1) transgenic is random knockout is specific
2) transgenic is multiple copies and knockout is a single
3) transgenic is a fertilised oocyte and knockout is a blastocyst
what are 3 characteristics of a HAC?
1) self replicating and self segregating
2) behaves as a stable chromosome
3) independent from the chromosomes of host cells
why are Talens and ZFNs difficult to design?
they rely on protein/DNA interaction
why is CRISPR/CAS9 easy to design and what can it be used for?
uses RNA to bind DNA - process bacteria use to protect themselves against viruses can be used for: 1) gene knock out 2) gene repression/ activation 3) gene alteration 4) gene insertion
what do ZN finger binder proteins do?
bind to a specific bit of DNA they recognise and cleave it and then bolt on to nucleases which can join sections of DNA together
what is a disadvantage fo ZN fingers?
Work out which combination of zinc fingers would bind specific bits of DNA - Had to create huge Zn libraries – reference against target DNA to identify correct place
how does CRISPR work?
complementary RNA sequences to specific part of genome can be genetically engineered by changing guide RNA to match the target- CAS 9 protein carries the targeting sequence and nuclease which cuts the DNA in the correct place
