genetic modification of animals Flashcards
define genetic modification
deliberate and targeted modification of the genetic makeup of an organism
what are the uses of genetic modification?
research, crops and livestock, industrial biotechnology, improving health, reproduction
what are the types of mutation?
substitution: one base switched to another
insertion: one base inserted into the gene
deletion: where a nucleotide or fragment is removed
what is nonhomologous end joining?
a repair process in which two double stranded breaks are fused directly with one another
what causes double stranded breaks?
- deletions caused by NHEJ
- irradiation
how can mutation frequency be enhanced?
- exposure to X rays
- exposure to DNA damaging chemicals such as procarbazine, methynitrosurea and ethylnitrosurea
how does ethylnitrosurea (ENV) cause mutations?
causes mutations in offspring of INV injected male mice crossed with female mice. does this by modifying DNA to cause mispairing at replication
what is a transgenic animal?
an animal in which an extra piece of DNA is introduced
what are transposons?
DNA elements used for insertional mutagenesis, work by inactivating the gene
why does homology dependent repair result in a greater range of mutations that can be inserted?
results in precise induction of mutations
outline non-homologous end joining
ends of double-stranded break processed by nuclease -> ends ligated with ligase -> the ds break is repaired, but there is a deletion where the break was
outline homology dependent repair
5’ ends of ds break processed with nuclease -> homologous recombination occurs using other chromatid as a template. can result in loss of heterozygosity
what are zinc fingers?
proteins that bind DNA, they have a characteristic 2 cysteines on one side, 2 Histidines on the other and a co-ordinated Zn ion in the middle
what is a ZFN?
a chimeric protein where a nuclease domain from the FOK1 protein has been added to 3 zinc fingers
why do you need two nuclease domains for a zinc finger nuclease to generate a DSB?
FOK1 nuclease domain only cuts one strand
after ZFN cleavage, how can you mutate a gene?
- add donor DNA homologous to the ends of DSB, initiating HDR, and targeted gene replacement
- allow NHEJ to occur, results in targeted mutagenesis
what are TALENs?
chimeric proteins consisting of a TALE domain (transcription activator-like effector) and a nuclease domain.
How do TALENs work?
relies on a repeat sequence and 2 variable AAs between each 35 AA block of repeats. allows binding with high specificity
outline the CRISPR/Cas9 system
- the bacterial sequence is transcribed into a long RNA with hundreds of repeats. tracrRNA is transcribed from a different place to create an RNA of 70-80 nts in length with region of homology w long RNA
- tracrRNA binds to long RNA, and RNase III cuts them off to create small complexes
- Cas9, in complex with tracrRNA and crRNA, bind DNA, Cas9 cuts DNA
how does the CRISPR/Cas9 system of gene editing differ from ZFNs and TALENs?
uses RNA to guide to the right sequence, rather than protein
what is the T7 endonuclease method for genotyping?
after a DSB, add 2 primers and amplify the mutated and wild-type allele. denature and re-anneal product. T7 endonuclease will cut ssDNA mutant fragments. if a deletion is present, there will be a mismatch in number of molecules, and a wild type band will be present
what methods of genotyping can you use?
- T7 endonuclease
- sequencing
- restriction assays
- southern blotting
briefly explain how you would design a CRISPR project
- compare gene of interest between mouse and human (identify the mutation you want to make)
- for homologous recombination, we want to cut close to mutation. use software such as CRISPOR to search for appropriate crRNAs with minimal off-target effects
- order the crRNA
- incubate crRNA, tracrRNA at 50 degrees for 30 mins, add Cas9 -> ribonucleotide complex forms
- inject into embryos or use electroporation
- add oligonucleotide of choice for homology dependent repair
what are the advantages of CRISPR?
- cheap
- quick
- easy
- applicable to all species
- diverse range of mutations
- applicable to any sequence in the genome
what are the off-target effects of CRISPR?
-some sequences other than the target will have homology, despite lacking appropriate PAM, some will cut
