Bresnick Lab

Tim’s cell media: What does beta-estrodial do?

• Contains estrogen

• Induces HOXB8-(activate) so Maintains progenitor cell at that state so it doesn’t differentiate

• Promoter of HOXB8 requires betaestrodiale to bind to HOXB8 into the plasmid so the plasmid

In cell culture what does HOXB8 do to the progenitor cell?

• HOXB8 is a transcription factor that keeps cells inactive and dividing without differentiation

• HOXB8 activates genes that mainatin progenitor identity and blocks differentiation

What will happen if Beta-estradiol doesn’t exist in HOXB?

• HOXB8 will be inactive

• causing cells to differentiate-grow from progenitor cells

How exactly does beta estrodial and HOXB8 come together

• Bind in the ER, causing HOXB8 to become active which can then block differentiation and cells stay in progentor state

What is G418

• antibiotic is inserted via virus

• bottom line cells with the plasmid survive

• always on-we want to kill any cells that lost the plasmid during profiletartion

• Plasmid contains promoter + HOXB8 sequence

plasmid also has promoter

What happens if inflammatory signal is left on?

Leads to immune diseases

What does GATA2 do? decommissioning of enhancers: A mechanism to constrain inflammatory signaling

• turns inflammatory signals of

Crispr Cas-9: What 3 components are involved?

1) tracRNA

2) cRNA

3) Cas-9 protein

What does the crRNA do

• Recognizes target DNA by base pairing

• tells Cas9 where to go

what does tracrRNA do

• binds to crRNA

• Help form a stable rna structure

What does cryo preserve do

stops cells from decay-preseves them

RPM isn’t atandardized among centrifuge machine-use GForce when using

• 300 Gforce

• when u have tube add FBS and media first BEOFRE DMSO (diluted) since otherwise cells will die

• 100% DMSO is toxic-that’s why add last after that

• DMSO prevents cell death by persevering bc of DMSO goes inside the cell and reduce ice crystal formation

• then resuspend cells in the tube

• then you put into Styrofoam for slow freezing (-80 fridge) to further reduce ice crystal formation

• 24-48 hours before removing otherwise tube wouldnt have been slow freeze to -80 before transferring to an even colder

• to prevent ice crystal formation

Mr.Frosty has ispropnyl

slows down the temp jump in the crypro bc of it’s feature of slowing down heat release low temp freezing point

• Beta estradiol binds to the promoter region of HOXB8 which is a transcription factor

• Now the binding of the promoter will activate promotor turning on the HOXB8 gene expression

Primitive Hemeopt.

Happens right at the yok sack- embryo, generates blood cells

• Don’t make long term HSC

What happens if you remove the -77kB enhancer? So higher receptors of IL6st and IL6RA and cytokine receptors increase

Leads to low GATA2 and higher inflammatory signal

• the enhancer regulates fir Gata2 transcription

What does CUT and Tag measure?

Measures how much protein has binded at that site Ex: where GATA2 binds to DNA

Secindary peroxide-color to where its bidng on secondary

How to dethaw cells?

• Take it out-warm the tube in hot water

• Put media (= media in the cryo) to wash the DMSO