基因表达调控-3

基因表达与调控
研究基因转录调控的方法
1. Reporter gene assay 2. EMSA (Electrophoretic mobility shift assay) 3. Foot-pringting 4. Methylation assay 5. Tet/off and Tet/on assay
Re:
Resolvase – to relaxation of twists in DNA helix
TetA: Actively expel intracellular tetracycline out of the cell
TRE:
Tetracycline-response element in the promoter of tetA 5`-TCCCTATCAGTGATAGAGA-3` , bind with TetR
Bert Vogelstein
Tet-Off/Tet-On
Tetracycline (Doxycycine) controlled gene expression system
Background:
Gene Structure of Tn10 Operon
TetR: repressor for tetA, can bind with Tetracycline T: Transposase – critical for operon jumping
rTetR(1-207Aa)—VP16 C-terminal activating domain (127Aa)
TRE in retrovirus plasmid pRev/TRE
Response virus
7 X TRE
tTA in pRevTet-off
rtTA in pRevTet-on
Regulation virus
6. Northern Blotting
7. ChIP 8. SAGE 9. Western blotting assay
Chromatin Immunoprecipitation (CHIP)
---- real-time analysis for protein/DNA interaction
You must know or have; 1. DNA sequence around interesting bind sites 2. Proteins that could bind to the sites 3. Antibody for the protein
9 bp DNA tag contains sufficient information to uniquely identify a mRNA
NNNN NNNNN NNNNNN NNNNNNN 44=256 45=1024 46=4096 47=16376
NNNNNNNN
NNNNNNNNN
48=65504
7. Determining effects of expression of interest gene on whatever you want to study.
PT67 Package Cells
3 different viral particles
Tet-off/Tet-on protocols
3. 4. Collect viral particles from culture medium If Tet-off:
Infect target cells by : pRevTet-off viral + pRevTRE-X viral;
14.
Design PCR primers that flack the binding site (200~500bp)
15.
Rea-time PCR
Serial Analysis of Gene Expression (SAGE)
----分析特定细胞或组织中的基因表达谱
SAGE is based on the following principle:
Select cells by G418/Hyg; Culture cells in the medium with doxycycline;
Before experiment, remove doxycycline.
Tet-off/Tet-on protocols
5. If Tet-on: Infect target cells by : pRevTet-on viral + pRevTRE-X viral;
7. Cut PCR product by Nla III and purify di-tag
8. Ligate to concatenation
9. Clone into Sph1 site in pSL301
NNNNNNGCATGCNNNNNNNN NNNNNNCGTACGNNNNNNNN 10. DNA sequencing
Target gene
Tet-off/Tet-on protocols
2. Transfect pRevTRE-X, pRevTet-off /pRevTet-on into packaging cells (PT67) separately
Response virus and regulate virus lack viral gene gag, pol and env that can be supplied by packaging cells
49=262016
BsmF1:
GGGACNNNNNNNNNNNNNNNNNNNN CCCTGNNNNNNNNNNNNNNNNNNNN
NlaIII:
NNNNCATGNNNNN NNNNGTACNNNNN
Streptavidin: A protein produced by the bacterium Streptomyces avidinii. It has four binding sites for biotin. It has been used extensively as probes in immunochemical systems, conjugated to antibodies, enzymes or fluorochromes.l II at transcription start point
---Nucleic Acids Research, 2004, 32(11): 1-8
1.
2.
Obtain tissues from animal or human
Crosslink chromatin and binding proteins Treat tissues by 1% formaldehyde for 12 min and then stop reaction by 0.125M Glycine
Select cells by G418/Hyg;
Culture cells in the medium without doxycycline; Before experiment, add doxycycline.
6.
Determining expression of target gene: Assay for mRNA --- PCR, Northern blotting for Protein -- enzyme activity, Western blotting
8.
Incubate sonicated DNA with blocked proteinA/G agarose to remove non-specific bind, spin to get supernatant, save sample as ―Input‖ Incubate pre-cleaned supernatant with antiRNA Pol II antibody
9.
10. Add blocked protein A/G agarose into reaction
11. Spin to collect protein A/G agarose and wash
12. Elute chromatin DNA by 1%SDS, 100mM NaHSO3
13. Incubate to reverse crosslink and use the sample as PCR temperate
1. Synthesize double strand cDNA from mRNA used biotinylated Oligo dT primer
2. Cut cDNA by Nla III and most 3-end portion was isolated by binding to streptavidin beads
3. divide cDNA in half (A and B)
4. Ligate adaptor A or B to each half of cDNA
5. Cut DNA by BsmF1 and blunt ends T4 DNA polymerase
6. Ligate A and B and amplify ditag by PCR with adaptor spercific primers
Tetracycline
Doxycycline
Tetracycline
Doxycycline
Tet-off/Tet-on protocols
1. Make response retrovirus pRevTRE-X Clone your gene of interest into pRevTRE
tTA:
Tetracycline-controlled Trans-Activator a recombinant protein by TetR(1-207Aa)—VP16 C-terminal activating domain (127Aa)