Lab 4: using RNAi and CRISPR/Cas9 to control gene expression

Question 1

What is RNAs major role?

Answer 1

• transmitting gene messages via mRNA to produce proteins.
• regulation of RNA - transcription, splicing, mRNA decay, and translation are regulated by protein factors and non-coding small RNAs.

Question 2

micro-RNAs (miRNAs)

Answer 2

• regulate gene expression by interferring with translation triggering decay of key mRNAs.
• observed in plants in early 1990s as co-suppression.
• In 1998, defined in C. elegans as “RNA interference” RNAi.

Question 3

RNAi

Answer 3

• double-stranded RNA (dsRNA), introduced into cells interferes with gene expression.
• leads to mRNA decay.
• technique based on this endogenous mechanism, used for gene knockdown.
• can be used as treatment for diseases like cancer, viral infections, and autoimmune diseases.
• RNAi is cheaper, fast, easier than traditional gene knockdown methods.

Question 4

Rescue experiments

Answer 4

• to confirm specificity of RNAi by restoring the protein function (eg., returning protein level to normal).

Question 5

Limitations of RNAi

Answer 5

Partial knockdown:
- RNAi reduces but does not completely eliminate gene expression so conclusions might be inconclusive if residual expression remains.

Question 6

CRISPR-Cas9

Answer 6

• new technology that can generate knockouts by introducing DNA double-stranded breaks using sgRNAs & Cas9.
• repairs by endogenous repair mechanisms often result in a gene KO (non-functional protein).
• advantage over RNAi cause CRISPR can completely remove gene expression.

Question 7

How was protein expression analysis done?

Answer 7

Western Blotting
- use antibodies to detect specific proteins on membrane.
- secondary antibody conjugated to HRP enzyme allows detection via chemiluminescence.
- use to assess protein levels and validate gene expression.

Question 8

ATGL

Answer 8

• Adipose Triglyceride Lipase
• Crucial in hydrolyzing triglycerides to release fatty acids and diglycerides regulating lipid homeostasis.
• ATLG dysfunction is linked to diseases like neural lipid storage diseases and various cancers.

Question 9

What is the set-up of this experiment?

Answer 9

• 4 HeLa Cell Pellets, transfected with:
  1) ShRNA (targeting ATGL knockdown - RNAi)
  2) CRISPR-Cas9 sgRNA (knockout)
• non-silencing shRNA & empty vectore CRISPR as controls.

Question 10

Why is puromycin used?

Answer 10

For selection of transfected cells.

Question 11

What is the procedure?

Answer 11

• prepare cell extracts - lyse cells with lsyis buffer.
• protein quantification.
• SDS-page & western blot.
• SDS-page is used to separate proteins by size, transfer to a membrane.
• then block with milk/TBST, incubate with primary antibodies (anti-ATGL & anti-B-actin), wash, & add secondary antibody.
• visualize bands with ECL (chemiluminescence)
• normalize ATGL expression to B-actin (loading control) to determine knockout/knockdown efficiency.

Question 12

What was used to stain lipid droplets in cells?

Answer 12

• Immunofluorescence: BODIPY DYE

Question 13

What is the goal of this experiment?

Answer 13

The goal is to determine the effect of ATGL knockdown (shRNA) or knockout (CRISPR) on lipids droplets in HeLa cells using western blotting & immunofluorescence to look at protein levels and lipid droplet accumulation.

Question 14

What is the purpose of skim milk used in the western blot?

Answer 14

• it is used as a blocking reagent.
• increases specificity of antibody binding to target protein, reducing background noise in final detection.

Question 15

What is the purpose of TBST?

Answer 15

• washing buffer
• help clean membrane without disrupting the antigen-antibody interactions, making it easier to detect the target protein.

Question 16

What was done in Week 1 of this experiment?

Answer 16

• the samples are (A (control for 1), 1, B (control for 2), 2.
  1) Prepare cell lysis buffer.
  2) Resuspend pellets.
  3) Centrifuge.
  4) Bradford assay/protein concentration determination.
  5) SDS.
  6) Western Blot.
  7) Ponceau Staining.

Question 17

What was done in week 2?

Answer 17

• wash membrane with TBST 3X = removes unbound antibodies to reduce non-specific background.
• can visualize the secondary antibody that is attached to first antibody using HRP (horseradish peroxidase) (do washing steps after 1st and 2nd antibody)
• ECL interacts with HRP to produce a luminescent signal.

Question 18

What is the purpose of actin?

Answer 18

Serves as a loading control for normalization. (it is bigger than ATGL so will not travel as fast on SDS page)

Question 19

What should the western blot results show us?

Answer 19

1) Loading control (B-actin) - bands appear similar intensity across all samples.

2) ATGL expression.
1. weaker band compared to the controls, indicating partial reduction in ATGL expression (knockdown)
2. no detectable ATGL band, indicating complete loss of ATGL expression (KO).

1. increased lipid droplet number area compared to control (A) indicating reduced ATGL expression. Slight increase in lipid number.
2. Large increase in lipid droplet area compared to control (B) due to complete ATGL loss. Slight or no increase in lipid number.