L12

Question 1

DNA cloning

Answer 1

• Can make a particular cell type express a gene they do not have or do not normally express by inserting a DNA fragment of interest into a plasmid vector

Question 2

DNA cloning steps

Answer 2

1. Need DNA of gene of interest (make copies via PCR)
2. Insert gene of interest into a plasmid vector (restriction enzyme is used to cut the ends of the DNA insert and the vector, DNA ligase links the DNA insert and vector together)
3. Bacterial transformation

Question 3

Genetic engineering in animals

Answer 3

• to investigate the effect of a particular gene and its product in physiological or pathophysiological conditions
  Example: is IRPV1 involved in the chronic pain mouse model of colitis
• measure abdominal pain in mice during colitis and after remission
• compare pain in different WT strain mice expressing TRPV1 vs not
• All WT mice trains express TRPV1
• Use genetically engineered mice that do not express TRPV1

Question 4

Knockout (KO) mice

Answer 4

Gene targeting: targeted gene is deleted or DNA sequence is inserted into TRPV1 gene to disrupt its expression
step 1: generate embryonic stem cells with recombinant DNA (DNA cloning but with mutated inactive version of TRPV1 gene)
step 2: transferring ES cell carrying targeted mutation into mice embryos
step 3: KO mice are identified and bred to produce mice of known genotype

Question 5

CRISPR-Cas9 genome editing

Answer 5

Clustered Regularly Interspaced Short Palindromic Repeats

• Involves 2 components: cas0 protein and single guiding DNA (20-nucleotide sequence complementary to the specific genomic site)
• Double-strand break, DNA repairs at the site via nonhomologous ending joining (small insertion, substitution or small deletion)