Chapter 35: Imaging Protein–Protein Interactions in Living Animals

Ramasamy Paulmurugan, Pritha Ray, Abhijit De, Carmel T. Chan, and Sanjiv Sam Gambhir

Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford University, Stanford, California 94305

ADDITIONAL PROTOCOLS

Protocol 3: Split Renilla luciferase complementation assay using rapamycin-mediated interaction of FRB and FKBP12; preliminary experiments in cell culture.

Protocol 4: Split Renilla luciferase complementation assay to study rapamycin-mediated interaction of FRB and FKBP12, using cell implants in living animals.

Protocol 5: Yeast two-hybrid strategy for imaging interaction of proteins X and Y in living cells.

Protocol 6: Yeast two-hybrid strategy for imaging interaction of proteins X and Y in living mice.

Protocol 3: Split Renilla Luciferase Complementation Assay Using Rapamycin-mediated Interaction of FRB and FKBP12; Preliminary Experiments in Cell Culture

For this protocol and Protocol 4, it is possible to substitute a combination of rapamycin, FRB, and FKBP12 with any other case in which a drug mediates a protein interaction.

The general strategies of transfection, cell harvest, and assay parallel those in Chapter 35 Protocol 1, with minor differences. It is useful to read and contrast the two protocols.

MATERIALS

Caution: See Appendix for appropriate handling of materials marked with <!>.

Buffers and Reagents

• 0.05 m Sodium phosphate <!> buffer (pH 7.0)
• Luciferase assay reagent (Promega)
• Passive lysis buffer (1×) (Promega)
• Phosphate-buffered saline (PBS) (pH 7.0)
• Bradford protein assay reagent (Bio-Rad)
• Superfect transfection reagent (Qiagen)
• Whole medium plus serum (DMEM + 10% FBS, 1% penicillin and streptomycin <!>)
• Coelenterazine (1 mg/ml in methanol) (Biotium, Hayward, CA)
• Rapamycin (1 mg/ml in dimethylsulfoxide) (Sigma)

Plasmid Vectors and Animal Cells

• 293T cells
• pcDNA3.1-NhRluc
• pcDNA3.1-ChRluc
• pcDNA3.1-NhRluc-FRB
• pcDNA3.1-FKBP12-ChRluc
• pcDNA3.1-hRluc
• pcDNA3.1-Fluc

The vector backbone pcDNA3.1(+) was from Invitrogen, and the Renilla luciferase gene fragments (NhRluc and ChRluc) were polymerase chain reaction (PCR)-amplified using the phRluc-CMV vector of Promega as template, and subcloned into pcDNA3.1. The control vector pcDNA-Fluc was constructed from the pGL3 vector (Promega). The DNAs encoding interacting proteins FRB and FKBP12 were PCR-amplified using pC₄-RE and pC₄M-FE, respectively, as templates and subcloned into the appropriate vectors.

Other Materials and Equipment

• Microfuge tubes
• Luminometer tubes
• Luminometer (Turner 20/20)

METHODS

1. On day 1, plate 1.5 ×10⁵ to 2 × 10⁵ 293T cells/well in 12-well culture plates and incubate at 37°C with 5% CO₂ for 24 hours. After 24 hours, cells will be about 80% confluent and ready for transfection.

2. On day 2, make transfection mixtures by adding the following reagents in the order listed:

1. Mock

   pcDNA3.1	400 ng/well
   pcDNA3.1-Fluc	10 ng/well
   Serum-free medium	75 μl/well
   Superfect	5 μl/well

2. Negative controls

i.	pcDNA3.1-NhRluc	200 ng/well
	pcDNA3.1	200 ng/well
	pcDNA3.1-Fluc	10 ng/well
	Serum-free medium	75 μl/well
	Superfect	5 μl/well
ii.	pcDNA3.1	200 ng/well
	pcDNA3.1-ChRluc	200 ng/well
	pcDNA3.1-Fluc	10 ng/well
	Serum-free medium	75 μl/well
	Superfect	5 μl/well
iii.	pcDNA3.1-NhRluc	200 ng/well
	pcDNA3.1-ChRluc	200 ng/well
	pcDNA3.1-Fluc	10 ng/well
	Serum-free medium	75 μl/well
	Superfect	5 μl/well
iv.	pcDNA3.1-NhRluc-FRB	200 ng/well
	pcDNA3.1	200 ng/well
	pcDNA3.1-Fluc	10 ng/well
	Serum-free medium	75 μl/well
	Superfect	5 μl/well
v.	pcDNA3.1	200 ng/well
	pcDNA3.1-FKBP12-ChRluc	200 ng/well
	pcDNA3.1-Fluc	10 ng/well
	Serum-free medium	75 μl/well
	Superfect	5 μl/well

3. Positive controls

   pcDNA3.1-hRluc	200 ng/well
   pcDNA3.1	200 ng/well
   pcDNA3.1-Fluc	10 ng/well
   Serum-free medium	75 μl/well
   Superfect	5 μl/well

4. Experiment

   pcDNA3.1-NhRluc-FRB	200 ng/well
   pcDNA3.1-FKBP12-ChRluc	200 ng/well
   pcDNA3.1-Fluc	10 ng/well
   Serum-free medium	75 μl/well
   Superfect	5 μl/well

For each of the eight samples (Experiment and Controls), make enough mix to transfect six wells (48 total) to allow assay of activity plus and minus rapamycin in triplicate. Incubate the transfection mixes for 20 minutes at room temperature.

3. During this time, wash the cells to be transfected once gently with PBS. Add 0.4 ml of fresh whole medium plus serum, and then add the transfection mix. Swirl gently to mix, and incubate for 3 hours at 37°C with 5% CO₂. Aspirate the medium to remove the excess transfection complex, and wash once more, carefully, with PBS.

4. To each set of the eight transfections (six wells per set), add 1 ml of fresh whole medium plus serum plus rapamycin (20 nm final concentration) to three wells of cells, and only fresh whole medium to the other three wells. Incubate the cells for 24 hours, and assay for normalized Renilla luciferase activity, using the protocol described below.

Renilla Luciferase Assay

1. On day 3, aspirate the medium for each well, and lyse the cells in 200 μl of passive lysis buffer, as described in Protocol 1.

2. To perform the hRluc assay, transfer 20 μl of lysate to a luminometer tube. Add 100 μl of sodium phosphate buffer containing 1 μg coelenterazine to the 20 μl of sample. Mix by vortex mixer, and count immediately using a luminometer for 10 seconds with a delay time of 2 seconds.

3. As described in Protocol 1, use another 20 μl of sample to measure the protein concentration, using a Bradford assay.

4. To normalize the transfection efficiency, assay the activity of the cotransfected firefly luciferase, using the assay described in Protocol 1.

5. Calculate the normalized RLU/μg protein/min, using the following formula:

   hRluc RLU in 20 μl of sample × hRluc dilution factor × 6
   ------------------------------------------------------------------------------------------------
   Total amount of protein in 20 μl of original sample × Fluc RLU in 20 μl
   of sample × Fluc dilution factor
    

Protocol 4: Split Renilla Luciferase Complementation Assay to Study Rapamycin-mediated Interaction of FRB and FKBP12, Using Cell Implants in Living Animals

This protocol closely parallels Protocol 2: Specific differences are noted.

MATERIALS

Caution: See Appendix for appropriate handling of materials marked with <!>.

Buffers and Reagents

• Coelenterazine (1 mg/ml in methanol)
• Phosphate buffered saline (PBS) (pH 7.0)
• Superfect transfection reagent (Qiagen)
• Whole medium plus serum (DMEM with 10% FBS, 1% penicillin and streptomycin <!>)
• Serum-free DMEM
• Rapamycin (1 mg/ml in dimethylsulfoxide)

Plasmid Vector and Animal Cells

• 293T cells
• pcDNA3.1
• pcDNA3.1-NhRluc-FRB
• pcDNA3.1-FKBP12-ChRluc

The vector backbone pcDNA3.1 (+) was from Invitrogen, and the Renilla luciferase gene fragments (NhRluc and ChRluc) were polymerase chain reaction (PCR)-amplified using the phRluc-CMV vector of Promega as template and subcloned into pcDNA3.1. The DNAs encoding the interacting proteins FRB and FKBP12 were PCR-amplified using pC₄-RE and pC₄M-FE as templates, respectively, and subcloned into the appropriate vector.

Other Materials

• Microfuge tubes
• Anesthesia (ketamine/xylazine)
• Mouse (strain nu/nu)

Equipment

• A cooled CCD (charged-coupled device) camera (Xenogen IVIS, Xenogen Corp.), in a light-tight temperature-controlled chamber

• Living Image Software (Xenogen Corporation) and Igor Image Analysis Software (Wavemetrics)

METHODS

1. To set up cells for transfection, follow instructions for Step 1, Protocol 2.

2. Make transfection mixes of the following reagents, adding components in the order as listed

1. Mock

   pcDNA3.1	5 μg
   Serum-free medium	500 μl
   Superfect	50 μl

2. Negative controls

   i.	pcDNA3.1-NhRluc-FRB	2.5 μg
   	pcDNA3.1	2.5 μg
   	Serum-free medium	500 μl
   	Superfect	50 μl
   ii.	pcDNA3.1-FKBP12-ChRluc	2.5 μg
   	pcDNA3.1	2.5 μg
   	Serum-free medium	500 μl
   	Superfect	50 μl

No positive controls can be studied in the same animals in this experiment. It can be done in a separate set of animals with either full reporter proteins or split reporter with known interacting partners.

3. Experiment

   pcDNA3.1-NhRluc-FRB	2.5 μg
   pcDNA3.1-FKBP12-ChRluc	2.5 μg
   Serum Free Medium	500 μl
   Superfect	50 μl

   Incubate transfection mixes for 20 minutes at room temperature.

3. Follow instructions for Step 3, Protocol 2, except here, dilute cells to 10 × 10⁶/100 μl in PBS, at the end of the procedure.

Optical CCD Imaging of Cell Implants in Living Mice

At least 12 mice will be required for this study.

1. To image rapamycin-mediated hRluc complementation in living mice, implant 10 ×  10⁶ 293T cells containing each of the four transfection mixes ([A] pcDNA3.1; [B] pcDNA3.1-NhRluc-FRB; [C] pcDNA3.1-FKBP12-ChRluc; [D] pcDNA3.1-NhRluc-FRB + pcDNA3.1-FKBP12-ChRluc, respectively) subcutaneously in four different zones (sites A, B, C, and D, respectively; see Fig. 3 of Chapter 35 of the second edition) of the anesthetized animal.

2. Beginning at time zero (= immediately after implantation), every 24 hours for up to 7 days, subcutaneously inject 50 μl of DMSO vehicle into one group of at least six animals as controls, and then inject 50 μl of 1 mg/ml rapamycin in DMSO into a second group of six animals. Next, inject all animals with 25 μg of coelenterazine intravenously, and then move immediately to imaging.

3. Anesthetize the animals by intraperitoneal injection of 40 μl of a ketamine and xylazine (4:1) solution.

4. Place the animals prone or supine on the temperature-controlled platform inside a light-tight chamber and obtain a gray scale reference image under low-level illumination.

5. Using a cooled CCD camera operated by Living Image software, collect photons emitted from cells implanted in the mice for a period of 1 minute.

6. To quantify the measured light, draw regions of interest over the area of the implanted cells and obtain the maximum photons/sec/cm²/steradian (sr), using Igor Image Analysis Software.

Protocol 5: Yeast Two-hybrid Strategy for Imaging Interaction of Proteins X and Y in Living Cells

Caution: See Appendix for appropriate handling of materials marked with <!>.

MATERIALS

Buffers and Reagents

• 0.05 m Sodium phosphate buffer (pH 7.0) <!>
• LARII (Luciferase assay reagent) (Promega)
• Passive lysis buffer (1×) (Promega)
• Phosphate-buffered saline (PBS) (pH 7.0)
• Bradford protein assay reagent (Bio Rad)
• Superfect transfection reagent (Qiagen)
• MEM with 10% FBS, 1% penicillin and streptomycin <!>
• Luciferin (30 mg/ml in PBS) (Xenogen Corp.)
• TNF-α (50 μg/ml in PBS) (Sigma)

Plasmid Vectors and Animal Cells

• 293T cells
• pcDNA3.1-NF-κB-Gal4-X
• pcDNA3.1-NF-κB-VP16-Y
• pcDNA3.1-Gal4-X
• pcDNA3.1-VP16-Y
• pcDNA3.1-(Gal4bs)₅-fl
• pcDNA3.1-Gal4-Z

pcDNA3.1-Gal4-X, pcDNA3.1-VP16-Y, and pcDNA3.1-(Gal4bs)₅-fl vectors were obtained from the CheckMate^TM Mammalian two-hybrid kit of Promega. pcDNA3.1-Gal4-Z was constructed by replacing the DNA-encoding protein X (interacting) with that encoding Z (noninteracting). pcDNA3.1-NF-κB-Gal4-X and pcDNA-NF-κB-VP16-Y were constructed by replacing the cytomegalovirus (CMV) promoter with the NF-κB promoter, polymerase chain reaction (PCR)-amplified from the template pNF-κB-Luc of Stratagene.

Other Materials and Equipment

• Microfuge tubes
• Luminometer tubes
• Luminometer (Turner 20/20)

METHODS

1. Twenty-four hours before transfection, split 293T cells to 0.5 × 10⁵ cells in each well of 12-well plates in whole medium plus serum and incubate at 37°C with 5% CO₂.

   Cells will be 80% confluent after 24 hours and ready for transfection. Since each transfection is performed in triplicate, to evaluate the five controls and one experimental interaction described below, 18 wells will be required, with 3 additional wells for each additional experimental interaction tested.

2. On day 2, make triplicate transfection mixes in microfuge tubes containing the following reagents. Add the reagents in the order listed.

   1. Mock

      pcDNA3.1-(Gal4bs)5-fl	1.5 μg/well
      Serum-free medium	75 μl/well
      Superfect	5 μl/well

   2. Negative controls

      i.	pcDNA3.1-Gal4-Z	0.5 μg/well
      	pcDNA3.1 -VP16-Y	0.5 μg/well
      	pcDNA3.1-(Gal4bs)5-fl	0.5 μg/well
      	Serum-free medium	75 μl/well
      	Superfect	5 μl/well
      ii.	pcDNA3.1-Gal4-X	0.5 μg/well
      	pcDNA3.1-NF-κB-VP16-Y	0.5 μg/well
      	pcDNA3.1-(Gal4bs)5-fl	0.5 μg/well
      	Serum-free medium	75 μl/well
      	Superfect	5 μl/well
      iii.	pcDNA3.1-NF-κB-Gal4-X	0.5 μg/well
      	pcDNA3.1-NF-κB-VP16-Y	0.5 μg/well
      	pcDNA3.1-(Gal4bs)5-fl	0.5 μg/well
      	Serum-free medium	75 μl/well
      	Superfect	5 μl/well

   3. Positive control

      pcDNA3.1-Gal4-X	0.5 μg/well
      pcDNA3.1-VP16-Y	0.5 μg/well
      pcDNA3.1-(Gal4bs)5-fl	0.5 μg/well
      Serum-free medium	75 μl/well
      Superfect	5 μl/well

   4. Experiment

      pcDNA3.1-NF-κB-Gal4-X	0.5 μg/well
      pcDNA3.1-NF-κB-VP16-Y	0.5 μg/well
      pcDNA3.1-(Gal4bs)5-fl	0.5 μg/well
      Serum-free medium	75 μl/well
      Superfect	5 μl/well

3. Incubate the transfection mixes for 20 minutes at room temperature.

4. During this time, wash the cells to be transfected once gently with PBS. Add 0.4 ml of fresh whole medium plus serum and then add the transfection mix. Swirl gently to mix, and incubate for 3 hours at 37°C with 5% CO₂.

5. Three hours following transfection, add TNF-α to the medium at a concentration of 0.05 μg/ml to all wells. Incubate cells for 24 hours.

Firefly Luciferase Assay

1. On day 3, aspirate the medium and lyse the cells in 200 μl of passive lysis buffer as described in Protocol 3. Remove 20 μl to assay protein concentration by Bradford assay and 20 μl for determination of normalized Fluc activity.

2. To assay Fluc activity, add 100 μl of LARII solution to 20 μl of sample. Mix by vortex mixer and count immediately in a luminometer for 10 seconds with a delay time of 2 seconds.

3. Estimate the normalized RLU in RLU/μg protein/min, as described previously.

Transfection normalization is difficult with this experimental setup. Hence, we used only proper positive and negative controls.

Dose–Response Study

1. To optimize NF-κB induction by TNF-α, it is advisable to optimize the TNF-α dose.

2. In triplicate, transfect 293T cells with the above-mentioned conditions and plasmids, and then add different concentrations of TNF-α  (0, 0.005, 0.01, 0.05, 0.1 μg/ml) to the media immediately after transfection.

3. After 24 hours, assay the cells for luciferase activity as described above.

Time Kinetics Study

1. It is also useful to optimize the time from TNF-α stimulation to reporter assay.

2. Transfect 293T cells with the above-mentioned plasmids, with a fixed concentration of TNF-α (0.05 μg/ml, or whatever is optimal in the previous dose-response study).

3. Assay the cells for Fluc activity as described above, after varying times of exposure (0, 6, 8, 24 hours).

Protocol 6: Yeast Two-hybrid Strategy for Imaging Interaction of Proteins X and Y in Living Mice

MATERIALS

Caution: See Appendix for appropriate handling of materials marked with <!>.

Buffers and Reagents

• 0.05 m Sodium phosphate buffer (pH 7.0) <!>
• LARII (Luciferase assay reagent) (Promega)
• Passive lysis buffer (1×) (Promega)
• Phosphate-buffered solution (PBS) (pH 7.0)
• Bradford protein assay reagent (Bio-Rad)
• Superfect transfection reagent (Qiagen)
• Whole medium plus serum (DMEM + 10% FBS, 1% penicillin and streptomycin <!>)
• Luciferin (30 mg/ml in PBS) (Xenogen Corp.)
• TNF-α (50 μg/ml in PBS) (Sigma)

Plasmid Vectors and Animal Cells

• 293T cells
• pcDNA3.1-NF-κB-Gal4-X
• pcDNA3.1-NF-κB-VP16-Y
• pcDNA3.1-Gal4-X
• pcDNA3.1-VP16-Y
• pcDNA3.1-(Gal4bs)₅-fl
• pcDNA3.1-Gal4-Z

pcDNA3.1-Gal4-X, pcDNA3.1-VP16-Y, and pcDNA3.1-(Gal4bs)₅-fl vectors were obtained from CheckMate^TM Mammalian two-hybrid kit of Promega. pcDNA3.1-Gal4-Z was constructed by replacing the DNA-encoding protein X (interacting) with that encoding Z (noninteracting). PcDNA3.1-NF-κB-Gal4-X and pcDNA-NF-κB-VP16-Y were constructed by replacing the cytomegalovirus (CMV) promoter with the NF-κB promoter, amplified by polymerase chain reaction (PCR) using pNF-κB-Luc of Stratagene as template.

Other Materials and Equipment

• Microfuge tubes
• Anesthesia (ketamine/xylazine)
• Mouse (strain nu/nu)

Equipment

• A cooled CCD (charge-coupled device) camera (Xenogen IVIS, Xenogen Corp.) in a light-tight temperature-controlled chamber

• Living Image Software (Xenogen Corporation) and Igor Image Analysis Software (Wavemetrics)

METHODS

1. On day 1, split 293T cells 24 hours before transfection to 3.0 × 10⁶ cells in each of nine 10-cm dishes in whole medium plus serum and incubate at 37°C with 5% CO₂. A total of nine plates each with 3.0 × 10⁶ cells for three sets of mice (1.0 × 10⁶ cells/mouse).

   Four mice each for mock and negative controls and eight mice for experimental group (nine plates of cells and 16 mice will be required for this experiment).

2. On day 2, make transfection mixes as follows, adding reagents in the order listed. Make each mix in triplicate:

1. Mock

   pcDNA3.1-(Gal4bs)5-fl	5 μg/well
   Serum-free medium	75 μl/well
   Superfect	μl/well

2. Negative control

   pcDNA3.1-Gal4-Z	1.75 μg/well
   pcDNA3.1-VP16-Y	1.75 μg/well
   pcDNA3.1-(Gal4bs)5-fl	1.75 μg/well
   Serum-free medium	75 μl/well
   Superfect	5 μl/well

   No positive controls can be studied in the same animals in this experiment. It can be done in a separate set of animals with only full reporter protein.

3. Experiment

   pcDNA3.1-NF-κB-Gal4-X	1.75 μg/well
   pcDNA3.1-NF-κB-VP16-Y	1.75 μg/well
   pcDNA3.1-(Gal4bs)5-fl	1.75 μg/well
   Serum-free medium	75 μl/well
   Superfect	5 μl/well

   Incubate mixes for 20 minutes at room temperature.

3. Wash cells gently one time with PBS. Add 2.5 ml of fresh whole medium plus serum to each transfection mix. Mix gently and add the mix to the cells. Incubate for 3 hours at 37°C with 5% CO₂. Aspirate the medium to remove the excess complex, and wash once carefully with PBS. Trypsinize and count the cells. Adjust the samples to 1.0 × 10⁶ cells/100 μl in PBS.

Optical CCD Imaging of Cell Implants in Living Mice

1. Anaesthetize the animals by intraperitoneal injection of 40 μl of a ketamine and xylazine (4:1) solution. Implant 1.0 × 10⁶ cells subcutaneously in mock, negative, and experimental groups.

2. Fifteen minutes after injection of the cells, inject 100 μl of d-Luciferin (30 mg/ml) intraperitoneally. Wait 5 minutes, and then proceed to imaging.

3. Place the animals prone or supine in a light-tight chamber and obtain a gray scale reference image under low-level illumination. Collect photons emitted from cells implanted in the mice for a period of 1 minute, with a cooled CCD camera, operated by Living Image Software.

4. After the first scan, inject each mouse in the induced experimental group, mock group, and control group with 0.5 μg of TNF-α  in PBS intraperitoneally and the uninduced experimental group with an equal volume of PBS.

5. Image all of these animals repetitively 8, 22, 30, and 48 hours later, after anesthetizing and injecting with 100 μl of d-Luciferin intraperitoneally.

6. Each time after scanning, re-inject the mice with another dose of TNF-α. To quantify the collected light, draw regions of interest over the area of the implanted cells and obtain the maximum photons/sec/cm²/steradian (sr), using Igor Image Analysis Software.

   Mice of experimental group induced with TNF-α should show increasing Fluc expression over 30 hours. The uninduced group should show increasing Fluc signal, but significantly lower than the induced group. Mice implanted with mock-transfected cells will show a very low level of Fluc signal.

APPENDIX

Sodium phosphate, NaH2PO4/Na2HPO4/Na3PO4, is an irritant to the eyes and skin. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety goggles. Do not breathe the dust.

Streptomycin is toxic and a suspected carcinogen and mutagen. It may cause allergic reactions. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses.