Lecture 4: Methods in Cell Physiology Part 2

CRISPR/Cas9 Gene Editing step 1

gRNA guides Cas9 to target sequence in genome

CRISPR/Cas9 Gene Editing step 2

Cas9 creates a double-stranded break in the DNA

CRISPR/Cas9 Gene Editing step 3

cell attempts to repair break

once DNA is cut, how will the cell repair directly

using nonhomologous end joining (NHEJ)

nonhomologous end joining

error-prone, generally inserts or deletes nucleotides

frame shift mutations often disrupt the gene downstream leading to what

gene knockout

what will the cell initiate by inserting an overlapping ~100 nucleotide single-stranded HDR template

homology-directed repair (HDR)

HDR template

possesses a small modification we want to introduce

what does HDR template leads to

gene knock-in

what does CRISPR interference (CRISPRi) target

the promoter

why does CRISPR interference (CRISPRi) target the promoter

prevents RNA polymerase from binding, blocks transcription of gene without permanently altering it

what are the other applications of CRISPR that use a defective Cas9 to transiently alter expression of a target gene

CRISPR interference, CRISPR activation

defective Cas9

dCas9

what do other applications of CRISPR that use defective Cas9 do

transiently alter expression of a target gene

what does CRISPR activation (CRISPRa) target

a region upstream of a promoter

how does CRISPR activation (CRISPRa) work

dCas9 fuses to VP16

what is VP16

a powerful transcription activator

how does VP16 work

drives expression of the gene of interest

how does RNA interference (RNAi) work

a way to transiently repress gene expression by preventing translation using antisense

a gene of interest is normally expressed in the sense direction, but what is it in RNAi

antisense

antisense

a transcript that enables complementary base pairing with the sense transcript

what does antisense transcript that enables the complementary base pairing with sense transcript form

dsRNA

how does the cell cleave dsRNA

using dicer

what does the cell cleaving dsRNA form

small interfering RNAs (siRNA)

what are siRNA used for

bind and degrade matching mRNA silencing the gene

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SDS-PAGE

what is SDS-PAGE

a technique used to separate proteins by mass

what is SDS

negatively-charged detergent that denatures proteins and coats them with a negative charge

how is SDS-PAGE set up

protein samples are loaded on a polyacrylamide gel and an electric current is applied

what happens to the protein in SDS-PAGE

migrate through pores in gel as the positive electrode attaches it

what size protein will migrate through pores faster

small

antibodies

Y-shaped proteins produced by the immune system

antibodies function

identify and neutralize antigens

antigens

foreign molecules

antibodies feature

highly specific to their antigen making them a powerful tool in cell physiology research and therapeutics

epitope

region of the antigen that the antibody recognizes

type of antibodies

polyclonal, monoclonal

polyclonal antibodies

mix of all antibodies the body produces against a particular antigen, will bind to different epitopes

monoclonal antibodies

purification of one antibody that binds to one specific epitope

how are polyclonal antibodies produced

by the immune system when a foreign antigen is introduced into the body

how does polyclonal antibodies production work

the purified protein of interest is injected into an animal and its immune system generates antibodies against it, a serum is collected

serum

collected from the animal’s blood in polyclonal antibodies production that contains all of the antibodies produced against the protein of interest

an example of purified polyclonal antibodies taken from the serum of immunized animals

antivenom

what does vemons mainly consist of

proteins, peptides

how can venoms be neutralized

by antibodies

large animals are vaccinated with venom to generate an antibody in response for what

emergency therapy

how are monoclonal antibodies produced

injects protein of interest into a mouse, mouse produces immune response against antigen, make hybridomas, selective media ensures only hybridoma cells survive, cells are screen individually for antibodies to antigen

What are the fused B cell–myeloma cells called

hybridomas

What is the first step in producing monoclonal antibodies

Introducing the protein (antigen) of interest into a mouse

What happens in the mouse after the antigen is introduced

The mouse produces an immune response against the antigen

Which cells are harvested from the mouse to make monoclonal antibodies

B cells

Why are mouse B cells fused with myeloma cells

To make the antibody producing cells immortal

Why is selective media used in monoclonal antibody production

To ensure only hybridoma cells survive

How are hybridoma cells tested after selection

They are screened individually for antibodies that bind the antigen

example of monoclonal antibody therapy

Adalimumab (Humira)

what does Adalimumab (Humira) do

specifically targets TNF to bind and neutralize it in the body alleviating inflammation

Tumor Necrosis Factor (TNF)

pro-inflammatory protein

what does Tumor Necrosis Factor (TNF) contributes to

rheumatoid arthritis, Crohn’s disease, Ulcerative Colitis, Plaque Psoriasis

how does SDS-PAGE separate proteins by

mass

is SDS-PAGE enough to confirm identity of protein of interest

no

immunoblotting/western blotting

uses antibodies specific to your protein of interest to label and visualize the presence of your protein

how does immunoblotting work

after separating proteins by mass with SDS-PAGE, it transfers proteins to a membrane to incubate with antibodies

primary antibody

binds to protein of interest/antigen

secondary antibody

binds to primary antibody

how can primary and secondary antibodies be modified

with a fluorophore or enzyme

why would you need to modify a primary antibody

for direct detection of the antigen

what does secondary antibody target

a region shared between all antibodies from that species

why would you need to modify a secondary antibody

for indirect detection of the antigen

what proteins did immunoblot detect

Actin, NEDD4, IFITM3

actin in immunoblot result

should be the same in all cell, it’s a loading control

NEDD4 in immunoblot result

knocked out in lanes 2, 4, 5, but added back in by a vector in lane 5

IFITM3 in immunoblot result

increases in expression when NEDD4 is knocked out

primary antibodies examples in immunoblot result

anti-Actin, anti-NEDD4, anti-IFITM3

what is the enzyme-conjugated secondary antibody used for in immunoblot result

reveals the interaction as bands show

what is incubated in immunoblot result

all primary antibodies then enzyme-conjugated secondary antibody

what instrument uses antibodies to show the presence and localization of proteins

flurescence microscropy

can antibodies be polyclonal or monoclonal

both

can antigens be directly or indirectly labelled

both

what can immunoblot results show about a protein