研習報告與討論 Summer Research Lab: 中央研究院生醫所 R206 賴建勳老師 Summer student: 林珮婷

Introduction Zinc Finger Protein( 鋅指蛋白 ) ‧ There are large amounts of zinc finger proteins in cells. ‧ In mammalian, most of them act as transcription factor, and are not only related to the control of genes but also the development of cells.

Zinc finger Protein

KRAB 12 C2H2 fingers N-terminal C-terminal Bind 輔抑制因子 Bind DNA The sequence of Z40A overlap the sequence of GATA family! GATA-1 red blood cells GATA-3 T cells

G G C G A T A A G T A A Z40A GATA-1 In Vitro

Material&Method Z40A DNA Binding Site Mutation Z40A Protein Domain Mutation

Z40A DNA Binding Site Mutation

Insert: Oligo linker TAGATGGCGATAAGTAATGATC TACCGCTATTCATTACTAGATC Wild-type Mutant Rule : A  G G  A C  T T  C (12 nucleotides)ZDBS Point mutation

Insert

Vector

Vector + Insert Ligation Transformation PCR screen Sequence confirmation Mammalian cell Transfection assay DNA large preparation Luciferase Assay Cloning

Mammalian Transfection Method: Calcium-Phosphate Transfection Ca Ca 2+ Effector : PEBH PEBH-Z40A-HA(6rev) DNA: ZDBS oligo DNA puc119 PRLnull

Cell line:293T (2x10 5 /3.5cm plate)

Cell line:293 (2x10 5 /3.5cm plate)

Discussion 1.The point mutation of 6th, 8th, 9th, nucleotides of ZDBS is really had more influence than others.The result is corresponding to the experiment in vitro. 2.Different cells have different reaction with the mutant of ZDBS. 3. Why the result is opposite to our prediction is still a topic to research.

Z40A Protein Domain Mutation Changing the nucleotides of Z40A DNA to product the wrong amino acid, therefore the structure of zinc finger is destroyed. We want to research which mutation of domains of Z40A is more important for Z40A to bind the DNA

Mammalian cell Transfection assay Acc65I XbaI Gel shift (mutation) Cloning

Cell line:293 (2x10 5 /3.5cm plate)

Discussion From the result, we can think that he zinc fingers of Z40A from seventh to twelfth are all very important for it to bind DNA.

1. Why we choose the more complex way (blunt end of vector and of insert) to clone the mutants of ZDBS? Q&A

進實驗室要學什麼？ 實驗室的要求？ 1. 要用功 2. 不重犯相同錯誤兩次 減少錯誤