Chapter 2 Materials and Methods
2.1 Materials
2.1.6 Kits
Kit Source Catalog
number
Application
Gene-SpinTM 1-4-3 DNA extraction kit
Protech PT-DNA14 3XL-V2
DNA extraction, clean-up
NucleoBond PC100
Macherey-Nagel 740573 DNA extraction, Midi preparation
Coomassie PlusTM Kit
PIERCE 23236 Detect protein
concentration
2.1.8 Buffers and chemical reagents
2.1.8.1 For cell culture
1X PBS (pH7.4)
137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4 i EDTA-trysin
2.5g trypsin, 0.1 M EDTA (pH8.0) in 1L 1X PBS, pH=7.4, 0.2 μm filtered
Versene
0.2 g EDTA in 1L 1x PBS
2.1.8.2 For molecular biology
50X TAE buffer
48.4 g Tris base, 0.5 M EDTA (pH8.0) 20 ml, 11.42 ml acetic acid added double distilled water (ddH2O) to 200 ml
Solution I (pH8.0)
50 mM Tris-HCl (pH=8.0); 10 mM EDTA (pH= 8.0); RNase A (stock:
100 μg/ml, work: 10mg/ml)
Per 100ml: add 5ml 1M Tris-HCl and 10ml 0.1M EDTA in ddH2O, adjust total volume to 100ml, autoclave, and add 1ml RNase A.
Solution I store at 4°C.
Solution II
0.2 M NaOH, 1% (w/v) SDS
Per 100 ml: add 0.8 g NaOH and 10 ml 10% SDS in DDH2O, adjust total volume to 100ml
Solution III（pH5.1）
2.8M KOAc
Per 100 ml: add 27.48 g KOAc in ddH2O, adjust pH to 5.1 by glacial acetic acid, and then adjust total volume to 100 ml
2.1.8.3 For cell staining
BSA-phOx-FITC
The preparation of 100 mg BSA dissolved in 10 ml, 0.1 M sodium bicarbonate solution (stock concentration is 1 M, pH=9), 100 mg phOx dissolved in 100 ml DMSO, and 100 mg FITC dissolved in 10 ml DMSO. 326 μl phOx and 584 μl FITC added in 10 ml, 10 mg/ml BSA, and shook in the shaker at RT for 90 minutes (The molar ratio, BSA vs.
phOx vs. FITC =1:10:10, the overall process must avoid the light). 1 ml, 1 M glycine was added and shook at RT for 15 minutes in excess to stop the reaction. BSA-phOx-FITC was purified by G-25 column. The chemical concentration was detected by Coomassie PlusTM kit (The kit major detect protein concentration. This solution included BSA, so that this kit detects the concentration). The equal volume BSA-phOx-FITC dissolved in the equal volume glycerol and stored at −20°C.
PB buffer (0.1M, pH7.4)
10.9 g Na2HPO4, 3.2g NaH2PO4 added ddH2O until 1L, adjust the pH of buffer to 7.4
phOx solution
100 mg phOx dissolved in 1ml DMSO
phOx-bead (phOx:bead=1:10, excess reaction)
100 μl magnetic beads, 5.43 μl phOx (1μg/μl) and 15 μg EDC mixed each other at 37°C for 4 hours. The PB buffer was added in this solution and purified by BD IMag™ Cell Separation System.
phOx-glycine (phOx:bead=1:10, excess reaction)
The preparation of glycine solution (10 mg glycine dissolved in 1ml 0.1 M sodium bicarbonate), get 1 ml glycine solution added in 289.6 μl phOx (100mg/ml) at 37°C for 4 hours
Staining buffer
1% BSA , 0.05% NaN3 in 1x PBS
2.1.9 Media
LB (Luria-Bertani) broth
1% tryptone, 0.5% yeast extract, 1% NaCl LB (Luria-Bertani)/Ampicillin broth
1% tryptone, 0.5% yeast extract, 1% NaCl, 50μg/ml ampicillin LB (Luria-Bertani)/Ampicillin agar
1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar, 50μg/ml ampicillin
SOB media
Per 1 L SOB media: 20 g Tryptone, 5 g Yeast Extracts, 0.5g NaCl and 0.186 g KCl (Before used, added 2.5 mL 2 M MgCl2 500 mL)
DMEM medium
13.35 g DMEM power, 3.7 g sodium bicarbonate in a glass cup, added ddH2O until 1L, adjust the pH of medium to 7.3
Growth medium with DMEM
10% FBS, 1% PSA in DMEM medium
5 mM polyethylenimine (PEI) Stock solution 1.25 g PEI in 10 ml ddH2O
2.1.10 Equipment
-20℃low temperature refrigerator (Frigidaire) 4℃ refrigerator (MINI KINGCON)
-80℃ low temperature refrigerator (NUAIRE) Bench top orbital shaker 060 (LMS)
Biophotometer DPU-414 (eppendorf) Centrifuge 5415D (eppendorf)
Centrifuge 5804 R (eppendorf)
DNA electrophoresis unit Gel Mate 2000 (Toyobo) Flow cytometer, FACSarray (BD)
Flow cytometer, FACScan (BD) Heating block (FIRSTEK)
Inverted research microscope, IX71 (Olympus) Laminar flow hood, Forma Class II, A 1284 (NSF) Microscope, CX31 (Olympus)
Orbital Shaking incubator OS1500R (TKS) pH meter SP701 (Suntex)
Thermal cycler (eppendorf)
Uni-photo gel image system (EZ lab) Water bath (FIRSTECK)
G-25 column (Bio-Rad, cat 7371532, USA)
The MidiMACS™ Separation System (Miltenyi Biotec, USA)
2.2 Methods
2.2.1 Construction of NF-κB based promoter library
2.2.1.1 Preparation of inserts (NF-κB based promoter library)
In due to create NF-κB based promoter library, NF-κB, CRE, MEF-2
and Sp1 binding sites are constructional materials in this construction. First, two complementary oligonucleotides (100 μM) were annealed to yield short DNA fragments that included individual binding sites at 95°C hot water and cooling down to room temperatureover 30 minutes. Next, these DNA fragments were phosphorylated for ligation, the reaction volumes as following:
Volume (μl) DNA kinase ATP Buffer ddH2O Total
NF-κB site 3 1 1 1 4 10
CRE site 3 1 1 1 4 10
MEF-2 site 3 1 1 1 4 10
Sp-1 site 3 1 1 1 4 10
Adaptor (Hind III cutting site)
3 1 1 1 4 10
These samples incubated at 37°C for 30 minutes. And then, different combination of NF-κB, CRE, MEF-2 and Sp1 were ligated in a total DNA volume of 6.4 µl using different volume ratio for creating more many combinations. The different DNA volume ratio and condition of ligation reaction listed in the following table.
A. The different DNA volume ratio P.S.: Each tube (L-1~L-5) the total DNA volume is fixed.
B. The condition of ligation reaction DNA
The ligation reaction incubated at room temperature (RT) overnight. These ligation products mixed together and ligated themselves (the product name:
LL-6). The condition of ligation reaction listed in the following table.
DNA
The ligation reaction incubated at RT overnight, and extracted DNA (the section 2.2.2) to condense volume. After the DNA extraction, DNA concentration was 114.8 ng/μl (total 3903.2 ng). Next, these DNA
fragments were combined with other elements (Hind III cutting sites, H’-1) by T4 ligase (2U). The condition of ligation reaction listed in the following table.
LL-6 H’-1 10 μM ATP ddH2O Buffer T4 ligase (2U) Total
34 μl 56 μl 13 μl 1 μl 13 μl 13 μl 130 μl
P.S.: The molar ratio between LL6 and H’-1 were 1:10.
The product was cleaned up by DNA extraction (the section 2.2.2) and DNA concentration was 105.3 ng/μl. After DNA extraction, the product was digested by Hind III restriction enzyme (MBI Fermentas, USA).
DNA (LL6+H’-1) Hind III ddH2O Buffer R Total
30 μl/ about 3 μg 1.5 μl 4.5 μl 4 μl 40 μl
P.S.: Hind III activity is 100% in Buffer R.
DNA (LL6+H’-1) concentration was 39.3 ng/μl. Next, these DNA fragments insert to vector with anti-phOx eB7 (the section 2.2.1.3).
2.2.1.2 Preparation of vectors (B16.4) and digestion by HindIII
Preparation of Plasmid DNA (B16.4) was used by Midipreparation kit (the section 2.2.4.1). B16.4 plasmid added HindIII and then digested. The digested condition was listed as following.
DNA (B16.4) HindIII ddH2O Buffer R Total
15 μl 10 μl 10 μl 65 μl 100 μl
The product was cleaned up by DNA extraction (the section 2.2.2) and DNA concentration was 477.8 ng/μl. After DNA extraction, the product was dephosphorylated to avoid self-ligation. The condition of dephosphorylated reaction listed in the following table.
DNA (B16.4) Phosphatase Buffer ddH2O Total
18 μl/6 μg 6 μl 3 μl 3 μl 30 μl
P.S.: Before ligation, the product was must heat- inactive in 10 minutes.
The B16.4 plasmids that digested by HindIII was inserted NF-κB based promoter library (LL6+H’-1). This process illustrated in the next section.
2.2.1.3 NF-κB based promoter library insert to vectors (B16.4)
The ligation reaction incubated at RT overnight and condition was listed in the following table.
Vector Insect Ligase (10U) ddH2O ATP Buffer Total
V+L 2.5 0 1.5 8 1.5 1.5 15
1:5 2.5 2 1.5 6 1.5 1.5 15
P.S.: The molar ratio between B16.4 and LL6+H’-1were 1:5
The products transformed later into electroporation competent cells (the section 2.2.3.3), and colonies numbers were listed in the following table.
Ligation condition Colonies number
V+L 100 1:5 2568
The total ligation products were transformed and incubated at 37°C for 8~12 hours in 100ml LB media (with kanamycin). No incubate for long times (>12 hours), because promoter library diversity maybe decrease.
2.2.2 DNA extraction (clean up)
The DNA digested or ligated by restriction enzyme or ligase was cleaned up by Gene-SpinTM 1-4-3 DNA extraction kit (Protech Taipei, Taiwan), following the commercial protocol. The DNA solution was spun at 13,000 rpm for 30 seconds in the spin column. The filtrate in the collection tube was discarded. 700 μl Washing solutions (Protech Co., Taipei, Taiwan) were added and the solution was spun for 1 minute at 13,000 rpm. This step was repeated twice. Then, the filtrate was discarded for 3 minutes at 13,000 rpm to remove residual trace of ethanol. The column was additionally incubated at 65°C for 5 minutes to evaporate ethanol. DNA was eluted by 30-50 μl ddH2O in a new tube.
2.2.3 Transformation of E. coli
Generally, heat-shocked competent cells were transformed and amplified plasmids except for plasmids with promoter library. The diversity of plasmids with promoter library is important, so that electroporation competent cells were used to transform them [transform efficiency: heat-shock (106~107) < electroporation (108~109)].
2.2.3.1 Preparation of competent cell (heat shock)
One pick of E. coli was inoculated in 3 ml of LB broth and grew for 12 hours at 37°C with vigorous shaking (~225 rpm). 1 ml of the overnight culture was transferred into 100 ml LB broth and was then incubated at 37
°C with shaking (~225 rpm) until the OD600 was between 0.35~0.45. The culture was stored on ice for 10 minutes. The cells were recovered by centrifugation at 4100 rpm for 10 min and then resuspended in 30 ml ice-cold 0.1 M CaCl2. The cells were pelleted by centrifugation at 4100 rpm for 10 minutes at 0°C. The pellet was resuspended in 2 ml 0.1 M CaCl2 (containing 10% glycerol). The cells were dispensed at 100 μl per eppendorf tube and then were stored at -80°C.
2.2.3.2 Transformation of competent cell (heat shock)
Stored competent cells were thawed on ice. 1 μl~2 μl of plasmid DNA was mixed with 100 μl competent cells and was then stored on ice for 30 minutes. The mixture was incubated in a preheated 42°C heating block for 90 seconds and quickly thawed on ice for 2 minutes. Then 250 μl of LB broth was added to the cells. The culture was incubated at 37°C with shaking (~225 rpm) for 50 minutes. 100 μl of the culture was plated on the LB agar plate with 50 μg/ml ampicillin or 30 μg/ml kanamycin. The plate was inverted and then incubated at 37°C for 12~18 hours.
2.2.3.3 Preparation of competent cell by electroporation
A colony of E. coli was inoculated in 25 ml of LB media and grew for
20 hours at 37°C with vigorous sharking (~225 rpm). The overnight culture was transferred into 500ml SOB containing 2 M MgCl2 and was then incubated at 37°C with shaking (~225 rpm) until the OD600 was between 0.35~0.45. The culture was stored on ice for 10 minutes. The cells were recovered by centrifugation at 2500 rpm for 15 minutes at 4°C and then resuspended was discarded. An appropriate amount of ddH2O was added to resuspend cells. The step was repeated twice. The pellet was resuspended in 1 ml ddH2O with 10% glycerol. The cells were dispensed at 20 μl per microtube and then were stored at -80°C.
2.2.3.4 Transformation of competent cell by electroporation
Stored competent cells were thawed on ice. 1 μl of plasmid DNA was mixed with 20 μl compement cells and transferred into a pre-cooled cuvette.
Cells was then electroporated at 2.5 mF, 2.5 kV, and 200 Ω for 4~5 milliseconds. The mixture was immediately recovered in 1 ml LB, transferred to a test tube, and incubated at 37°C with agitation (~225 rpm) for 1 hour, 100 μl of the culture was plated on the LB agar plated or 100 ml both in 250 ml flask with 50 or 30 μg/ml kanamycin respectively. The plate or flask was incubated at 37°C.
2.2.4 Plasmid DNA extraction
2.2.4.1 Minipreparation
A single colony of E. coli was inoculated in 3 ml of LB broth (with
antibiotics) and grew overnight at 37°C with vigorous shaking (~225 rpm).
1~2 ml of the cells were recovered by centrifugation at 13,000 rpm for 1 minutes and then resuspended in 200 μl ice-cold Solution I buffer in a new tube. 250 μl Solution II buffer was added and mixed gently, then stood at RT for 10 minutes. 250 μl Solution III buffer was added to the mixture and mixed gently, then stood on ice for 10 minutes. Cells were spun at 13,000 rpm for 5 minutes at 4°C. The supernatant transferred to a fresh tube. And then an equal volume of phenol: chloroform (~700 μl) was added. The organic and aqueous phases were mixed by vortexing and then centrifuge the emulsion at 13000 rpm for 3 minutes at 4°C in a tube. The aqueous upper layer transferred to a fresh tube. DNA was precipitated from the supernatant by adding 0.7 (0.6~1) volumes of isopropanol at RT. the solution was mixed completely and then allowed the mixture to stand for 2 minutes at RT. The precipitated DNA was collected by centrifugation at 13000 rpm for 20 minutes at 4°C. The supernatant was removed by gentle aspiration. The tube stood in an inverted position on a paper towel to allow all of the fluid to drain away. 1 ml of 70% ethanol added to the pellet and inverted the closed tube several times. The DNA recovered by centrifugation at 13000 rpm for 5 minutes at 4°C in tube. The tube opened at RT until the ethanol has evaporated and no fluid was visible in the tube (5~10 minutes). The DNA dissolved in 50 μl ddH2O and stored at -20°C.
2.2.4.2 Midipreparation
A single colony of E. coli was inoculated in 100 ml of LB broth (with antibiotics) and grew overnight at 37°C with vigorous shaking (~225 rpm).
The broth was centrifugated at 8,000 rpm at 4°C for 15 minutes. After supernatant was discarded, 4 ml buffer S1 (with RNase A) was added and then solution was vortexed. 4 ml buffer S2 was added to the suspension.
The lysate was mixed gently by inverting the tube 6~8 times and incubated at RT for 2~3 minutes (max 5 minutes). Do not vortex, as this will release contaminating chromosomal DNA from the cellular debris into the suspension. The solution was added pre-cooled 4 ml buffer S3 (4°C) and inverted gently 6~8 times until a homogeneous suspension containing an off-white flocculate was formed. The suspension was incubated on ice for 5 minutes. A NucleoBond AX 100 Midi column was equilibrated with 2.5 ml buffer N2. The flow-through was emptied by gravity flow and discarded.
The bacterial lysate was cleared by centrifugation at 12,000 rpm at 4°C.
The lysate was then loaded onto the NucleoBond column, which was emptied by gravity flow. 10 ml buffer N3 was added to wash the column.
This step was repeated once again. Plasmid was eluted with 5 ml of buffer N5 3.5 ml isopropanl was added to precipitate the eluted plasmid. The mixture was incubated on ice for 10 minutes and centrifuged at 13,000 rpm for 30 minutes at 4°C. 1 ml 70% ethanol was added to the pellet and stored at -20°C or the solution was centrifuged at 13,000 rpm for 5 minutes for further application. Last, the pellet was re-dissolved in 20 μl ddH2O.
2.2.5 Cell culture and Subculture
2.2.5.1 Cell culture
Balb/3T3, 293, B16F10 and A549 cells were mainintained 90%
DMEM, 10%FBS, 1%PSA medium and incubated at 37°C. These cells were subcultured 2~3 times in the week.
2.2.5.2 Subculture
Remove and discard culture medium. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed. Add 6.0 to 8.0 ml of growth medium and aspirate cells by gently pipetting. Add appropriate aliquots of the cell suspension to new culture vessels. These cells all were incubated at 37°C.
2.2.6 Polyethylenimine (PEI) transfection
Seeding 300,000 cells in 6-well 18hr before transfection (to make 50~70% confluency at the time of transfection). Immediately before transfection, cells were rinsed and supplemented with fresh serum-free culture medium (0.2 ml). The plasmid DNA (3~4 μg) and the desired amount of PEI were each diluted into 100 μl of 150 mM NaCl and vortexed.
The different amounts of PEI used in different cell lines. After 5 minutes, add the PEI solution into plasmid DNA solutions (Notice: not the reverse
order), and then were vortexed. After 20 minutes, washed the well and dropped 200 μl Opti-MEM, then the transfection mixture was added to the cells, and then add 600μl Opti-MEM. After 18 hours incubation, 2 ml growth medium were added into each well. After 24~48 hours, the gene expression level was analyzed by flow cytometry.
2.2.7 Stain transfecants
Discard the culture medium at 48 hours after transfection. Rinse each well with 1ml PBS. Add 1ml Versene and incubate it at 37°C for 5 minutes.
Add 1 ml DMEM into each well to harvest the transfectants. Centrifuge the cell mixture at 1500 rpm, at 4°C for 5 minutes. Discard the supernatant and suspend the pellet with 1ml staining buffer the cell into FACS tube.
Centrifuge the mixture at 1500 rpm, at 4°C for 5 minutes. Discard the supernatant and put the tube upside down for 30 seconds. Add 1 μl BSA- phOx-FITC or 1 μl magnetic bead/ml staining buffer 1 hour. Wash the pellet with 1ml staining buffer twice. Analysis the partial cells by staining 1 μl BSA-phOx-FITC with flow cytometry. The others isolated by magnetic beads separation system combined with competing agents (phOx-glycine).
2.2.8 The isolation of different activity promoters by MBSCS
The transfecants to be separated is first magnetically labeled with 1 μl phOx-bead. After magnetic labeling, the transfecants are suspended in 3
mL PBS and passed through an LS Separation Column that is placed in the strong permanent magnet of the MidiMACS Separation Unit. The magnetizable column matrix serves to create a high-gradient magnetic field.
The magnetically labeled cells are retained on the column and separated from the unlabeled cells that pass through until 3 ml solution is eluted. 3 ml competing agents (1 μl phOx-glycine dissolved in 3 ml PBS) were added.
After removal of the column from the magnetic field, added 3 mL PBS in LS Separation Column and the retained fraction can be eluted.
2.2.9 Extraction of plasmids by Hirt method
The cells from magnetic beads separation were pelled by centrifugation and resuspended in125 μl of phosphate-buffered saline (PBS). An equal volume of 2X Hirt buffer was added and the solution was incubated at room temperature for 15min to allow the cell to lyse.
After the additition of 62 μl of 5 M NaCl to give a final concertation of 1 M, the solution was placed at 4°C overnight. The samples were the centrifuged at 14000x g for 60 minutes at 4°C, and the suspentant was extracted with an equal volume of phenol-chloroform. The DNA was ethanol precipitated and resuspend in 10mM Tris-HCl, pH=7.4.
Chapter 3 Results
3.1. Polyethyleminie transfection assay
In order to establish the selectable system of promoter library, the transfection efficacy is important in this my experiment. DNA transfection technique was shown to deliver genes into various cell lines successfully, (Boussif, Zanta et al. 1996) its transfection efficiency is higher than other cationic polymers (Demeneix, Behr et al. 1998; Read, Singh et al. 2005).
In this experiment, Different doses of PEI/DNA complex were tried to transfect efficiently into various cell lines, including Balb/3T3 (murine fibroblast), 293 (human kidney cell), B16-F10 (murine melamona cell), A549 (human lung carcinoma cell), DBTRG-05MG (human glioblastoma cell) and HepG2 (human hepatocellular carcinoma cell) cell lines.
The efficiency rates of PEI transfection for Balb/3T3 and 293 cells were 64.95% and 60.92%, respectively (Figure 7). For A549 and B16-F10 cells, the efficiency rates were 26.58% and 25.84%, respectively (Figure 7, 8) However, (Figure 8), the efficiency rates only were 11.28% and 4.24%
for the DBTRG-05MG and HepG2 cells, respectively. Therefore, PEI transfections were easy for Balb/3T3, 293, A549 and B16-F10 cell lines.
PEI transfections were difficult for DBTRG-05MG and HepG2 cells.
Because PEI transfection for Balb/3T3, 293, A549 and B16-F10 cells were better transfection efficiency than HepG2 and DBTRG-05M, these
cells were selected as target cells . Furthermore, Balb/3T3 and 293 cells are belonging to immortal, non-tumorous. In contrast, A549 and B16-F10 cells are belonging to tumorous. They can be studied the genes expression between non-tumor and tumor cells.
3.2. Assay of NF-κB expression level in various cell lines
As previous description, NF-κB activities are higher in tumor cells than normal cells. Therefore, the NF-kB activities were monitored in Balb/3T3, 293, A549 and B16F10 cells to determine whether the difference exists in the selected target cell line.
In this experiment, pNF-κB–hrGFP vector was used to detect NF-κB activity in Balb/3T3, 293, A549 and B16F10 cell lines (Figure 9, 10). Two plasmids were co-transfected into target cells: one plasmid containing the TFBS which drove the green fluorescence protein (hrGFP) gene; the other containing CMV promoter which drove the red fluorescence protein (AsRed). Co-transfection with pAsRed-N2 could avoid the difference in transfection efficiency at different experiment groups. Moreover, pARE-hrGFP containing ARE site (prokaryotic TFBSs) was as negative control vector to normalize the degree of transgene expression in different cell lines. The expression folds formula is described as below:
CMV-AsRed X ARE-hrGFP TFBSs-hrGFP
Expression Fold =
In Balb/3T3 and B16F10 cells, pNF-κB-hrGFP expression was 12-fold and 34-fold than pARE-hrGFP, 30-fold and 12-fold than
pAP-1-hrGFP (Figure 9). In 293 and A549 cells, pNF-κB-hrGFP expression was 4-fold and 28-fold than pARE-hrGFP, 1.2-fold and 21-fold than pAP-1-hrGFP (Figure 10). The NF-κB activities were lower in normal-like (293 and Balb/3T3) cells than tumor cells (A549 and B16F10).
Interestingly, all cell lines had higher NF-κB activities than other transcription factors in this experiment (Figure 9, 10).
3.3. Construction and selection of NF-κB based promoter library
Promoters’ activity dependent to transcription factors and NF-κB has higher activity in tumor cells. Therefore, a new NF-κB based promoter library was created to select a new promoter sequence which can be overexpressed in tumor cell but not in normal cell.
3.3.1. Construction of NF-κB based promoter library
NF-κB based promoter library was created by randomly ligating oligonucleotides containing NF-κB, CRE, MEF-2 or Sp1 sites. First,two complementary oligonucleotides were synthesized for each individual
NF-κB based promoter library was created by randomly ligating oligonucleotides containing NF-κB, CRE, MEF-2 or Sp1 sites. First,two complementary oligonucleotides were synthesized for each individual