genetic modification of animals

Question 1

define genetic modification

Answer 1

deliberate and targeted modification of the genetic makeup of an organism

Question 2

what are the uses of genetic modification?

Answer 2

research, crops and livestock, industrial biotechnology, improving health, reproduction

Question 3

what are the types of mutation?

Answer 3

substitution: one base switched to another
insertion: one base inserted into the gene
deletion: where a nucleotide or fragment is removed

Question 4

what is nonhomologous end joining?

Answer 4

a repair process in which two double stranded breaks are fused directly with one another

Question 5

what causes double stranded breaks?

Answer 5

• deletions caused by NHEJ

- irradiation

Question 6

how can mutation frequency be enhanced?

Answer 6

• exposure to X rays

- exposure to DNA damaging chemicals such as procarbazine, methynitrosurea and ethylnitrosurea

Question 7

how does ethylnitrosurea (ENV) cause mutations?

Answer 7

causes mutations in offspring of INV injected male mice crossed with female mice. does this by modifying DNA to cause mispairing at replication

Question 8

what is a transgenic animal?

Answer 8

an animal in which an extra piece of DNA is introduced

Question 9

what are transposons?

Answer 9

DNA elements used for insertional mutagenesis, work by inactivating the gene

Question 10

why does homology dependent repair result in a greater range of mutations that can be inserted?

Answer 10

results in precise induction of mutations

Question 11

outline non-homologous end joining

Answer 11

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

Question 12

outline homology dependent repair

Answer 12

5’ ends of ds break processed with nuclease -> homologous recombination occurs using other chromatid as a template. can result in loss of heterozygosity

Question 13

what are zinc fingers?

Answer 13

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

Question 14

what is a ZFN?

Answer 14

a chimeric protein where a nuclease domain from the FOK1 protein has been added to 3 zinc fingers

Question 15

why do you need two nuclease domains for a zinc finger nuclease to generate a DSB?

Answer 15

FOK1 nuclease domain only cuts one strand

Question 16

after ZFN cleavage, how can you mutate a gene?

Answer 16

• add donor DNA homologous to the ends of DSB, initiating HDR, and targeted gene replacement
• allow NHEJ to occur, results in targeted mutagenesis

Question 17

what are TALENs?

Answer 17

chimeric proteins consisting of a TALE domain (transcription activator-like effector) and a nuclease domain.

Question 18

How do TALENs work?

Answer 18

relies on a repeat sequence and 2 variable AAs between each 35 AA block of repeats. allows binding with high specificity

Question 19

outline the CRISPR/Cas9 system

Answer 19

• 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

Question 20

how does the CRISPR/Cas9 system of gene editing differ from ZFNs and TALENs?

Answer 20

uses RNA to guide to the right sequence, rather than protein

Question 21

what is the T7 endonuclease method for genotyping?

Answer 21

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

Question 22

what methods of genotyping can you use?

Answer 22

• T7 endonuclease
• sequencing
• restriction assays
• southern blotting

Question 23

briefly explain how you would design a CRISPR project

Answer 23

• 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

Question 24

what are the advantages of CRISPR?

Answer 24

• cheap
• quick
• easy
• applicable to all species
• diverse range of mutations
• applicable to any sequence in the genome

Question 25

what are the off-target effects of CRISPR?

Answer 25

-some sequences other than the target will have homology, despite lacking appropriate PAM, some will cut