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研習報告與討論

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 901638. 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

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研習報告與討論

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  1. 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 901638

  2. 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.

  3. Zinc finger Protein

  4. N-terminal C-terminal 12 C2H2 fingers KRAB 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

  5. GATA-1 Z40A In Vitro • 2 3 4 5 6 7 8 9 10 11 12 • G G C G A T A A G T A A

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

  7. Z40A DNA Binding Site Mutation

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

  9. Insert

  10. Vector

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

  12. Mammalian Transfection Method: Calcium-Phosphate Transfection Effector : PEBH PEBH-Z40A-HA(6rev) DNA: ZDBS oligo DNA puc119 PRLnull Ca2+ Ca Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+

  13. Cell line:293T (2x105/3.5cm plate)

  14. (2x105/3.5cm plate) Cell line:293

  15. Discussion • 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. • 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.

  16. 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

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

  18. Cell line:293 (2x105/3.5cm plate)

  19. 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.

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

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

  22. 謝謝大家

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