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

IntroductionZinc 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 sequenceof GATA family!

GATA-1 red blood cellsGATA-3 T cells

1 2 3 4 5 6 7 8 9 10 11 12G 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 : AG GA CT TC

(12 nucleotides)ZDBS

Point mutation

Insert

Vector

Vector + InsertLigation

Transformation

PCR screen

Sequence confirmation

Mammalian cell Transfection assay

DNA large preparation

Luciferase Assay

Cloning

Mammalian Transfection

Method:

Calcium-Phosphate Transfection

CaCa2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Effector : PEBH PEBH-Z40A-HA(6rev)DNA: ZDBS oligo DNA puc119 PRLnull

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000

pEBH

pEBH

-Z40

A-H

A(6r

ev)

pEBH

pEBH

-Z40

A-H

A(6r

ev)

pEBH

pEBH

-Z40

A-H

A(6r

ev)

pEBH

pEBH

-Z40

A-H

A(6r

ev)

pEBH

pEBH

-Z40

A-H

A(6r

ev)

pEBH

pEBH

-Z40

A-H

A(6r

ev)

WT LS9 LS6 LS8 LS11 LS12

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

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

pEBH

pEBH-Z40A-HA(6rev)

pEBH

pEBH-Z40A-HA(6rev)

pEBH

pEBH-Z40A-HA(6rev)

pEBH

pEBH-Z40A-HA(6rev)

pEBH

pEBH-Z40A-HA(6rev)

pEBH

pEBH-Z40A-HA(6rev)

pEBH

pEBH-Z40A-HA(6rev)

pEBH

pEBH-Z40A-HA(6rev)

WT LS9 LS3 LS5 LS6 LS8 LS11 LS12

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

Discussion1. 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

Acc65IXbaI

Gel shift

(mutation)

Cloning

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

PEBH PEBH-

Z40A-

HA(6REV)

PEBH-

Zf(1--

>12)(6rev)

PEBH-

KRAB-

Zf(7--

>12)WT

PEBH-

KRAB-

Zf(7--

>12)7mut

PEBH-

KRAB-

Zf(7--

>12)8mut

PEBH-

KRAB-

Zf(7--

>12)9mut

PEBH-

KRAB-

Zf(7--

>12)10mut

PEBH-

KRAB-

Zf(7--

>12)11mut

PEBH-

KRAB-

Zf(7--

>12)12mut

Cell line:293(2x105/3.5cm 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