Yeast two hybrid screening of liver and WAT libraries

I may need 15 instead of 10 minutes.

Bait & prey ?

cDNA library inserted here

Library construction

• RNA reverse transcription

• PCR amplification of cDNA

• recombination of cDNA with pGADT7-rec vector

Library cDNA recombination in AH109 yeast

First screening

• Cotransformation of pAS2-1-PERK, cDNA and pGADT7-rec

• Media are deficient of Trp, Leu, Ade, His. ((But His is leaky.))

• AH109 yeast contains mutations in TRP1, LEU2, ADE2 and HIS3.

• AH109 has four reporters—ADE2/HIS3/MEL1/lacZ controlled by GAL4.

Re-streak to separate different plasmids

Big clones were picked and streaked 2-3 times on –Ade/-His/-Trp/-Leu media plus 2.5mM 3-AT.

The 4th streak was done on –Ade/-His/-Trp/-Leu media plus 7.5mM 3-AT.

PCR identification of clones

250 Yeast plasmids were amplified with PCR and sequenced

Liver clones

Liver clones

Liver clones

adaptor-related protein complex AP-1, mu subunit, ATX1 (antioxidant protein 1) homolog 1, BTB domain containing 9 (Btbd9), gamma-aminobutyric acid receptor associated protein (Gabarap)high mobility group 20 B (Hmg20b) microtubule-associated protein 1 light chain 3 beta (LC-3)Mus musculus polymerase, delta interacting protein 2 (Poldip2), protein inhibitor of activated STAT, 4 (Pias4), SNF related kinase (Snrk),

WAT clones

cell division cycle 5-like (S. pombe) (Cdc5l), Ewing sarcoma breakpoint region 1 (Ewsr1), G protein beta subunit-like (Gbl), GABA-A receptor-associated protein (Gabarap)HRAS like suppressor 3 (Hrasls3), leukotriene C4 synthase (Ltc4s), lipoprotein lipaseNuclear protein SkiP (Ski-interacting protein)profile/Regulator of chromosome condensation containing proteinRho GTPase activating protein 17 (Arhgap17), sex comb on midleg homolog 1 (Scmh1),

syntaxin 5A (Stx5a),

What’s next ?

Cotransformation of the postive PCR product back to AH109.

GST pulldown ?

Co-IP ?

Any one passes all the requirement will be further characterized in vivo.