Introduction B cell Bone marrow T cell Thymus
Introduction B cell （ Bone marrow） T cell（ Thymus） Lymphocyte stem cell （Bone marrow） Tc Th （Cytotxic T cell） （Helper T cell）
Introduction § Th 1 cells activate macrophages. Enabling them to destroy intracellular microorganisms more efficiently. § Th 2 cells drive B cells to differentiate and produce immunoglobulins of all other type.
Introduction T-bet Th 0 Th 1 IFN-γ
Material Yeast
Main difference between yeast and E. coli culture § Incubation time Yeast E. Coli 4~6 days 16 hours § Incubation temperature Yeast E. Coli 30℃ 37℃ § Culture medium Yeast E. Coli YPD LB
Method Yeast One Hybrid system
§ One-Hybrid System provides an in vivo genetic assay used for isolating novel genes encoding proteins that bind to a target, cis-acting regulatory element § Protein-DNA interaction
GAL 4 AD AD AD BD BD Constructed plasmid BD BD a. Fusion protein expression AD AD BD BD b. Fusion protein binding AD c. Activation AD BD BD 報導基因 Reporter gene Yeast chromosome IFN-r promoter T-bet
Eco. RI Reporter gene Xba. I IFN-r promoter ligation
Activation domain Eco. RI Pst. I T-bet binding domain Report gene ligation
Yeast strain：YM 4271（MATa）、Y 187（MATα） Can’t synthesis histidine、 leucine、uracil by itself。 Incubate Yeast selection medium Yeast -His or -Leu or -Ura Yeast can’t growth on selection medium
Yeast selection medium p. HISi-IFN-γ -His Yeast（ YM 4271） Yeast selection medium p. GAD 424 -T-bet Yeast（ Y 187） -Leu
3 -AT：a inhibitor of the HIS 3 gene product Appropriate 3 -AT Yeast selection medium p. HISi-IFN-γ Yeast（ YM 4271） -His
Yeast can’t growth Incubate p. HISi-IFN-γ Yeast selection medium Yeast（ YM 4271） -His/-Leu Yeast can’t growth Incubate p. GAD 424 -T-bet Yeast selection medium Yeast（Y 187） -His/-Leu
p. HISi-IFN-γ p. GAD 424 Appropriate 3 -AT Yeast selection medium -His/-Leu
Yeast mating YM 4271 Y 187 p. HISi-IFN-γ p. GAD 424 -T-bet Yeast selection medium -His/-Leu
YM 4271 Y 187
Future work § Screen library to find new transcription factor.
THE END Thank you
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