LAB 4

Question 1

ATGL

Answer 1

55kDa protein
rate limiting step in lipolysis of lipid droplets
visualised by BODIPY
cancer is often associated with altered metabolism, and that includes lipid metabolism
ATGL expression is altered in many cancers

Question 2

difference between knockdown and knockout

Answer 2

knockdown: suppression or reduction in gene expression
knockout: removal of genes from DNA which results in complete loss

Question 3

RNAi and regulatory pathway of miRNAs

Answer 3

Question 4

what is siRNA

Answer 4

Question 5

what is shRNA

Answer 5

Question 6

genome editing machinery

Answer 6

Question 7

what is non homologous end joining

Answer 7

endogenous RNA machinery places and takes away random bases to fill in the ds-break generated by Cas9
produces a premature stop codon -> KO

Question 8

what is homology directed repair (HDR)

Answer 8

a repair template contains the desired nucleotide change
precise gene editing -> knock ins (large insertion), deletions, mutations

Question 9

lab 4 week 1 protocol

Answer 9

Question 10

lab 5 week 2 protocol and results

Answer 10

beta actin is used as a control for protein expression

Question 11

why would one use EDTA instead of EGTA in a protein isolation?

Answer 11

Question 12

Answer 12

BODIPY- lipids
DAPI- nucleus

Question 13

what does CRISPR stand for

Answer 13

clustered regularly interspaced short palindromic repeats

Question 14

what does western blot do

Answer 14

assess level of protein expression
employs specificity of antibodies by exposing membrane to an antibody that recognises a specific protein
secondary antibody recognises primary antibody and is conjugated to horse radish peroxidase
add lightning reagent (ECL) and then visualise chemiluminescence

Question 15

what did the four HeLa cells pellets given contain

Answer 15

transfected with shRNA or a non silencing vector
or CRISPR Cas9 sgRNA targeting or CRISPR Cas9 empty vector
puromycin was used to select for transfected cells

Question 16

why is skim milk a good blocking buffer

Answer 16

prevents non specific binding
reduces background noise

Question 17

what is the mechanism of CRISPR Cas9

Answer 17

1. CRISPR array is transcribed into a precursor CRISPR derived RNA (pre-crRNA)
2. trans activating crRNA is transcribed
   it has homology with the repeat sequences
   recruits RNase III to process the pre-crRNA and complexes with Cas9
3. Cas9:crRNA complexes are formed
4. crRNA guides complex to target sequence, specificity comes from recognition of a short protospacer adjacent motif
5. DNA is unwound at target sequence
6. Cas9 makes a double stranded break at the target sequence

Question 18

RNAi and CRISPR Cas9 comparison

Answer 18