Experiments with tetracycline as a regulator of inducible gene expression
Experiments with tetracycline as a regulator of inducible gene expression
The following protocol is divided into 3 stages: stable transfection of fibroblasts with pTet-tTAk, stable transfection of NIH-3T3 cells inducible to express tTA, and analysis of protein expression in the transfected cells. Stably transfected cell lines expressing trans-activators and target genes were divided into two phases. This experiment was derived from the next volume of Molecular Cloning Laboratory Guide (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Experiments with tetracycline as a regulator of inducible gene expression
Materials and Instruments
pSV2-His pTet-Splice with open reading frames of target genes pTet-Spliqe with reporter genes NIH-3T3 cells pPGKPuro Stable cell lines inducible to express self-regulatory tTAs Move Stage 1: Stable transfection of fibroblasts with pTet-tTAK For more product details, please visit Aladdin Scientific website.
CaCl2 calf serum Chloroquine HEPES buffer Phosphate buffer Suitable restriction endonuclease Trypsin-EDTA DMEM Complete culture medium Selection medium Protein sample buffer Antibodies Tris-glycine SDS-polyacrylamide gel
Plastic cloning rings Polypropylene vinyl tubing Tissue culture dishes Tissue culture plates Boiling water baths Polyvinylidene fluoride membranes
Materials
Buffers and solutions
Dilute the stock solution to the appropriate concentration.
CaCl2 (2mol/L)
CaCl2(2mol/L) solution is filtered to remove bacteria and then dispensed in 5 ml portions per serving and stored at -20°C.
Calf Serum (10%)
Chloroquine (10 mg/ml)
Optional, see Step 7.
Chloroquine is dissolved in water and the solution is filtered and stored at -20°C.
10 mg/ml chloroquine is equivalent to 19 mmol/L. See the Chloroquine Diphosphate section of Chapter 16, Information Box.
Glycerol (15%) HEPES Buffer Dilution
HEPES Buffer
Phosphate buffer
Enzymes and buffers
Suitable restriction endonucleases
See step 2.
1X Trypsin-EDTA
0.05% Trypsin
0.5 mmol/LEDTA(PH8.0)
Culture solution
DMEM complete culture medium (Dulbecco's modified Eagle's medium complete)
DMEM
100 units/ml靑霉素
100ug/ml Streptomycin
2 mmol/L Glutamine
10% donor calf blood淸
DMEM complete culture solution with 0.5 tons/ml tetracycline-HCl
Tetracycline-HCl is dissolved in 70% ethanol at a concentration of 10 mg/ml and stored at -20°C. DMEM complete culture solution containing 0.5ug/ml tetracycline-HCl (with or without selective reagents) used in this protocol can be stored at 4°C, protected from light, for approximately 1 month.
Selective Culture Solution containing L-histidind and 0.5ug/ml tetracycline
Histidine-free DMEM
100 units/ml penicillin
100ug/ml Streptomycin
2 mmol/L glutoxamide
10% donor calf serum
125,250 or 500 umol/L L-histidinol (prepare 125 mmol/L stock solution for storage at -20°C)
0.5ug/ml tetracycline-HCl (dissolved in 70% ethanol at 10mg/ml, stored at -20°C)
Histidine-free DMEM is available from Irvine Scientific (Santa Ana,California) Selected cultures containing 0.5ug/ml tetracycline-HCl can be stored in the light at 4°C for approximately 1 month.
Specialty Equipment
Plastic cloning rings
Cloning rings are autoclaved vertically on a thin layer of vacuum grease.
Polypropylene tubes (4 ml)
Tissue culture dishes (6 cm and 10 cm)
These protocols can be scaled down so that fewer cells are needed, smaller petri dishes and plates are used, and all components are proportionally reduced.
Additional reagents
Step 23 requires the reagents listed in Chapter 7, Scheme 8 or Appendix 8.
Vectors and Strains
pSV2-His
plasmids were both purified by CaCl-ethidium bromide gradient equilibrium centrifugation (Scheme 10 in Chapter 1) or chromatography columns (Scheme 9 in Chapter 1).
For information on pSV2-Hia, see Damke et al. (1995). Plasmids pTet-tTAk and pTet-Splice are available from Life Technologies, as are other vectors with selection markers (e.g. pCI-neo from Promege, pTK.HYG from CLONTECH). If replacing pSV2-His with other vectors, use a selection medium suitable for the selection marker of the plasmid.
pTet-Splice with an open reading frame of the target gene
Optional, see step 2.
pTet-tTAk
Cells and tissues
Cultured mammalian cells
NIH-3T3 cells are used in this protocol, but other cells can certainly be used. Cells are grown in a suitable culture medium.
Methods
Culturing and transfecting cells
1. Cultivate adherent cells in DMEM complete culture medium. The day before transfection, change the cells into DMEM complete culture medium containing 0.5ug/ml tetracycline-HCl (tetracycline). Add enough cells to each 10 cm dish so that on the day of transfection, the cells can have 33% confluence.
IMPORTANT: From this point on, cells are cultured at 37°C, 5% CO2, in 0.5ug/ml tetracycline-HCl unless otherwise indicated.
2. Linearize the plasmids at the appropriate restriction endonuclease sites and adjust the concentration of each plasmid to ≥0.5 mg/ml. 10-20ug of plasmid pTet-tTAk and 1-2ug of pSV2-His (the molar ratio of Tet plasmid to plasmid with selective markers is approximately 10:1) are mixed with 500ul of HEPES buffer in a clean 4 ml polypropylene vinyl tube. tube.
If target genes are to be cotransfected, add as many pTet-Splice recombinants with target genes as there are moles of pTet-tTAk. 
3. 32.5ul of 2mol/L CaCl2 is added to the DNA mixture. immediately mix the solution by gentle shaking. The solution is kept at room temperature and mixed from time to time. a flocculent precipitate will form after 15-30 min.
For more details on transfection of cells with calcium phosphate, see Protocols 2 and 3 in Chapter 16.
4. Pipette off all the culture solution from the cell culture dish prepared in step 1.
5. Mix the CaCl2-DNA several times with a Pasteur pipette. Add the mixture dropwise so that it covers the monolayer of cells evenly.
6. Incubate the cells at 37°C, 5% CO2 for 30 min and shake the dish after 15 min to ensure uniform coverage of the DNA precipitate.
7. 10 ml of DMEM complete culture medium containing tetracycline was added to each cell culture dish. The cells were incubated at 37°C, 5% CO2 for 4-5 h.
If tolerated, a final concentration of 25umol/L can be added at this time. The optimal transfection incubation time for different cell types may vary.
8. Gently aspirate the culture solution. Avoid destroying the sediment on the cells.
9. Add 2.5 ml of HEPES buffer containing 15% glycerol and preheated to 37°C for glycerol shock. Cells are left at room temperature for 2.5 min. glycerol is added dropwise to the culture medium.
It is normal for the cells to look a little crumbly after glycerol shock. The use of chloroquine (step 7) will further reduce the integrity of the cells, but will improve transfection efficiency.
10. Aspirate off the glycerol solution at exactly 2.5 min. Do this quickly because glycerol is very toxic to cells.
For certain cell types that are highly resilient, the time the cells are in the glycerol solution can be increased to 4~5 min in order to optimize transfection efficiency.
As a general rule, the maximum time for cell shock should allow about 50% of the cells to survive.
11. Immediately wash the cells by gently and quickly adding 10 ml of DMEM complete culture medium containing tetracycline. Immediately aspirate the culture medium and repeat the wash.
IMPORTANT: Because the cells are easily dislodged from the culture dish after glycerol shock, the culture solution should all be added in one place.
12. Add 10 ml of DMEM complete culture medium containing tetracycline to the cells and incubate at 37°C overnight.
13. About 16~24 h after transfection, aspirate the culture medium and replace it with 10 ml of DMEM complete culture medium containing tetracycline.
After transfection, incubate at 37°C for a total of 48 h (i.e., the total incubation time of steps 12 and 13).
Screening and cloning of transfected cells
14. 48 h after transfection, cells were divided into culture medium containing 125umol/L histidinol and 0.5ug/ml tetracycline-HCl at several dilutions. Cell densities ranged from approximately 1X106 to 3X104 per 10 cm dish. several dishes containing approximately 1X105 cells were cultured.
15. Cells were cultured in selection medium for 4 days, followed by 3 to 4 ml of selection medium containing 125umol/L histidinol and 0.5ug/ml tetracycline-HCl.
16. After colony formation (usually after about 10-12 days of selective culture), the cells were switched to selective culture medium containing 250umol/L L-histidinol and 0.5ug/ml tetracycline-HCl.
L-Histidinol is usually toxic to cells. Therefore, the concentration of L-histidinol in the selection medium should be increased only when the amount of pSV2-His cells with high level of expression reaches the threshold value.
17. After the colonies are well grown (12~15 days of selection culture), draw the boundary of each colony with a circle at the bottom of the culture dish. Aspirate off the culture medium and place a sterile plastic cloning ring on the individual clones of the petri dish. Repeat this step for each picked colony. Picking cells from a petri dish requires that the individual colonies be relatively well separated and easily distinguishable.
Important: After stable transfection with pTet-tTAk, cells must be cultured in culture medium containing 0.5ug/ml tetracycline in order to prevent toxic effects of tTA expression and subsequent screening of tTA-overexpressing clones.
18. Wash the clones quickly with about 100ul of phosphate buffer. To dislodge the cells, add 2 drops of 1X trypsin-EDTA (~100ul) and leave for 30-60 s. Blow up and down with a Pasteur pipette to loosen the cells. Each colony was transferred to a well of a 24-well tissue culture plate with 1 ml of selection medium containing 250umol/LL histidinol and tetracycline in each well.
Subsequent cell passages were performed by standard methods, such as (1) rapid washing with PBS; (2) 1-3 min trypsin-EDTA digestion (2 ml per 10-cm cell-filled dish); and (3) dilution/termination of trypsin activity with tetracycline-containing Selection Medium or 10% calf's blood淸.
19. When the cells in the wells reach 80% confluence, they are transferred to 6 cm tissue culture dishes and incubated with a selection medium containing 500 umol/L L-histidinol and tetracycline.
NIH-3T3 cells can be grown to 80% confluence in approximately 4-7 days; however, the time required to grow to 80% confluence varies among cell lines and even among clones.
20. Cells are expanded in selection medium containing 500umol/L L-histidinol and tetracycline (typically cells are diluted 1:5 to 1:10).
Cells are tested for inducible protein expression
21. When the monolayer is grown again to approximately 80% confluence, a portion of each cell clone is collected and frozen in liquid nitrogen. The remaining cells are passaged and cultured until the number is sufficient for inducible expression of the test protein.
For future use of frozen cells, they are resuscitated and then cultured in DMEM containing 500umol/L L-histidinol and 0.5ug/ml tetracycline-HCl but no histidine.
22. If cells are cotransfected with tTA and the target plasmid, the product of the target gene can be analyzed directly as described in stage 3. If the cells are transfected with the pTet-tTAk plasmid only, the cells are tested for inducible expression as follows:
a. One day prior to induction, cells are cultured in DMEM culture medium containing 500umol/L L-histidinol and 0.5ug/ml tetracycline-HCl at an appropriate density so that they can reach a subconfluent state at the time of harvest. b. The cells are then incubated with tTA and target plasmid as follows.
b. On the second day, wash the cells three times with PBS, gently rotating the culture plate each time.
c. Immediately after the third wash, add selection medium containing 500umol/LL-histidinol but no tetracycline. Cells were cultured in culture medium with or without tetracycline for 6~48 h. The cells were incubated with PBS for 3 times, each time gently rotating the plate. 
23. Inducible expression of tTA in cells was tested by Northern analysis or immunoblotting. Cell lines expressing tTA were then transfected with the target plasmid as described in stage 2.
In stably transfected NIH-3T3 cells, expression of tTA and target genes is usually observed after 6 h of induction, with expression reaching a maximum after a further 6 h. The expression of tTA and target genes in stably transfected NIH-3T3 cells is usually observed after 6 h of induction.
Stage 2: Stable transfection of NIH-3T3 cells inducibly expressing tTA with tetracycline-regulated target genes
Materials
Buffers and solutions
Bovine serum (10%)
HEPES buffer
Phosphate buffer
Enzymes and buffers
Suitable restriction endonucleases
1X Trypsin-EDTA
0.05% Trypsin
0.5 mmol/LEDTA (pH 8.0)
Culture solution
Selective culture medium containing 500 umol/L-histidinol and 0.5ug/ml tetracycline (with or without 3ug/ml puromycin)
DMEM without histidinol
100 units/ml Cyanamycin
100ug/ml Streptomycin
2 mmol/L Glutamine
10% calf serum
500 umol/LL-histidinol (diluted from 125 mmol/L stock solution, stored at -20°C)
0.5ug/ml tetracycline-HC1 (dissolved in 70% ethanol at 10mg/ml, stored at -20°C.)
Histidine-free DMEM is available from Irvine Scientific (Santa Ana, Califomia) Selected cultures containing 0.5ug/ml tetracycline-HCl can be stored at 4°C, protected from light, for approximately 1 month.
Add 3ug/ml puromycin when appropriate.
Special equipment
Plastic cloning ring
Cloning rings are sterilized by autoclaving them vertically on a thin layer of their empty fat.
Polypropylene tubes (4 ml)
Tissue culture dishes (6 cm and 10 cm)
Tissue culture plates (24 wells)
Additional reagents
Step 3 of this phase requires the reagents listed in Phase 1 of this protocol.
Vectors and Strains
pPGKPuro (or vectors with other selection markers)
All plasmids were purified by CaCl-liquefied ethidium gradient equilibrium centrifugation (Chapter 1, Scheme 10) or chromatography columns (Chapter 1, Scheme 9).
For information on selective labeling, see Damke et al. (1995). Plasmids pTet-Splice and PUHC13-3 are available from Life Technologies. Other vectors with selection markers are also available (e.g. pCI-neo from Piomega, pTK-HVG or pPUR from CLONTECH). If replacing pPGKPuro with another vector, use a selection medium suitable for the selection marker of the plasmid.
pTet-Spliqe with reporter gene (optional, e.g. PUHC13-3)
pTet-Splice with open reading frame of target gene
Cells and tissues
Stable cell lines inducible to express self-regulated tTA (Phase 1)
Methods
Culture and transfection of cells
1. Cultivate a stable cell line (isolated in stage 1) that can be induced to express self-regulated tTA in complete selection medium containing 500umol/LL-histidinol and 0.5ug/ml tetracycline-HCl. One day before transfection, cells were passaged into 10 cm tissue culture dishes containing complete selection medium. A sufficient number of cells were transfected into each dish to allow 33% confluence of the monolayer on the day of transfection.
IMPORTANT: From this point on, cells are cultured at 37°C, 5% CO2, in 0.5ug/ml tetracycline-HCl unless otherwise indicated.
2. Linearize the plasmids at the appropriate restriction endonuclease site and adjust the concentration of each plasmid to ≥0.5 mg/ml. 10-20ug of each poop gene plasmid and 1-2ug of pPGKPuro (10:1 molar ratio of each tetracycline plasmid to plasmid with selective markers) are mixed with 500ul of HEPES buffer in a clean 4 ml polypropylene vinyl tube. 
3. Perform steps 3 to 13 in Stage 1.
IMPORTANT: Where Stage 1 called for DMEM complete culture medium containing tetracycline, be sure to switch to Selection Medium containing 500umol/L L-histidinol and 0.5ug/ml tetracycline-HCl for this transfection.
Selection and cloning of transfected cells
4. 48 h after transfection, cells were divided into selection medium containing 500umol/L L-histidinol, 3ug/ml puromycin and 0.5ug/ml tetracycline at several dilutions. Cell densities ranged from approximately 1X106 to 3X104 per 10 cm dish, and several dishes containing approximately 1X105 cells were required.
The lowest concentration of puromycin that will kill all untransfected cells within a few days needs to be determined empirically prior to transfection and varies with cell type. 3ug/ml puromycin is sufficient to screen transfected NIH-3T3 cells. Puromycin is effective in killing most cell types at a range of concentrations.
5. Cells were cultured in selection medium for 4 days followed by 3-4 ml of selection medium containing 500umol/L L-histidinol, 3ug/ml puromycin and 0.5ug/ml tetracycline.
6. After the colonies are well grown (12~14 days of selective culture), draw the boundary of each colony with a circle on the bottom of the Petri dish. Aspirate off the culture solution and place a sterile plastic cloning ring on the individual clones of the petri dish. Repeat this step for each selected colony. Picking cells from a petri dish requires that the individual colonies be relatively well separated and easily distinguishable.
7. Wash the clones quickly with approximately 100ul of phosphate buffer. To dislodge the cells, add 2 drops of 1X trypsin-EDTA (~100ul) and leave for 30-60s. Blow up and down with a Pasteur pipette to loosen the cells. Each colony was transferred to a well of a 24-well tissue culture plate with 1 ml of selection medium containing 500umol/L L-histidinol, 3ug/ml puromycin, and 0.5ug/ml tetracycline in each well.
Subsequent cell passages were performed by standard methods such as (1) rapid washing with PBS, (2) 1~3 min trypsin-EDTA digestion (2 ml for each cell-lined dish), and dilution/termination of trypsin activity with 10% calf serum (3 ml).
8. When the cells in the wells grew to 80% confluence, they were transferred to 6 cm tissue culture dishes and cultured with selection medium containing 500umol/L L-histidinol, 3ug/ml puromycin and 0.5ug/ml tetracycline.
NIH-3T3 cells can be grown to 80% confluence in about 4-7 days; however, the time required to grow to 80% confluence varies among cell lines and even among clones.
9. Cells are expanded in selection medium containing 500umol/LL-histidinol, 3ug/ml puromycin, and 0.5ug/ml tetracycline (usually cells are diluted 1:5 to 1:10).
10. When the monolayer of cells has grown again to approximately 80% confluence, a portion of each cell is collected by charging and frozen in liquid nitrogen. The remaining cells are passaged until sufficient numbers are available to test for inducible expression of target gene proteins. The test method is described in Stage 3.
For subsequent use of frozen cells, they are resuscitated and then cultured in a selection medium containing 500umol/LL-histidinol, 3ug/ml puromycin and 0.5ug/ml tetracycline-HCl.
Stage 3: Analysis of protein expression in transfected Tin cells
Materials
Buffers and solutions
Dilute the stock solution to the appropriate concentration.
Calf serum (10%)
Phosphate buffer
1X Protein Sample Buffer
Antibody
Antibodies suitable for the determination of target proteins of interest by immunoblotting.
Gel
Tris-Glycine SDS-Polyacrylamide Gel
The concentration of acrylamide used for gelling should be suitable for the observation of the target protein, see Appendix 8.
Culture solution
Selective culture medium containing 500umol/L L-histidinol, 3ug/ml puromycin (with or without 0.5ug/ml tetracycline)
DMEM without histidine
100 units/ml puromycin
100ug/ml Streptomycin
2 mmol/L Glutamine
10% calf's blood淸
500 umol/L L-histidinol (diluted from 125 mmol/L stock solution, stored at -20°C)
3ug/ml puromycin
Irvine Scientific (Santa Ana,California) sells histidine-free DMEM.
Tetracycline was added when appropriate to give a concentration of 0.5ug/ml (tetracycline-HCl dissolved in 70% ethanol at a concentration of 10 mg/ml and stored at -20°C). Selected cultures containing 0.5ug/ml tetracycline-HCl can be stored at 4°C, protected from light, for about 1 month.
Special Equipment
Boiling water bath
Polyvinylidene difluoride (PVDF) membranes
Additional reagents
Step 13 of this protocol requires the reagents and equipment listed in Appendix 8 for immunoblotting.
Cells and Tissues
Stable cell lines that can be induced to express self-regulated tTA and contain plasmids carrying target genes of interest
Methods
Cell growth and induction
1. The night before induction, cells were cultured in DMEM culture medium containing 500umol/L L-histidinol, 3ug/ml puromycin, and 0.5ug/ml tetracycline-HCl at an appropriate density so that they could reach a subconfluent state at the time of harvest.
2. On the second day, cells were washed 3 times with PBS, each time gently rotating the culture plate.
3. Immediately after the third wash, selective culture medium containing 500umol/L L-histidinol, 3ug/ml puromycin but no tetracycline was added. The cells were cultured in the culture medium with or without tetracycline for 6~48 h. The cells were then incubated for 6~48 h in the culture medium with or without tetracycline.
In stably transfected NIH-3T3 cells, expression of tTA and target genes was usually seen after 6 h of induction, with peak expression after about 12 h of induction. 
Harvested cells
4. After the cells have been grown for an appropriate period of time, they are quickly harvested and placed into tubes in an ice bath.
If cells are harvested with trypsin, wash the cells first with cold phosphate buffer free of calcium and magnesium salts, then terminate trypsin action with cold selective culture medium containing 10% calf's blood淸 (with or without tetracycline, as needed). The tubes were immediately transferred to an ice bucket.
5. 0. 5X106 cells are transferred to centrifuge tubes for each clone and control, and then all tubes are centrifuged at 3000 r/min (low or medium speed) for 5 min at 4°C.
6. Wash the cells with 1 ml of ice-cold phosphate buffer. Wash the cells as in step 5, gently aspirating off the supernatant without disturbing the cell sediment.
7. Place the cell sediment on an ice bath and loosen the sediment by swirling the tube briskly in the well above the centrifuge tube rack, then freeze it at -70°C.
Preparation of Lysates
8. The cell precipitate is resuspended with 30ul of Protein Sample Buffer, the cells are gently blown, and the tube is shaken.
This step may also be done before freezing the cells.
9. Cells are boiled in Protein Sample Buffer for 10 min.
10. Cell debris is collected by centrifugation at maximum speed for 2 min.
11. Add 10ul of cell lysate to each lane of a Tris-Glycine SDS-Polyacrylamide gel.
If the cell lysate is not added to the gel immediately, it can be stored at -20°C, but needs to be boiled again before sampling.
12. Run the gel for the appropriate amount of time (see Appendix 8).
13. Proteins are electrotransferred from the SDS-polyacrylamide gel onto a PVDF membrane and detected with a suitable antibody (please see Appendix 8). 
