Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

Summary

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Abstract

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Protocol

1. Before-you-start Technical Considerations

1. We recommend using both C57/BL6 wild-type mice and knockout mice for experiment because mice with corresponding CD molecules can be purchased from major mouse vendors.
2. Since it is difficult to track the wild-type mice and knockout mice derived donor cells after bone marrow transplantation, it is necessary to use CD45.1 mice to represent wild-type mice. (CD45.1 C57/SJL WT mice Jackson Laboratories stock #002014).
3. Most mouse colonies are CD45.2. Our knockout mice belong to CD45.2 genotype (alternatively a CD45.2 WT mice source: Jackson Laboratories stock #000664). The determination of CD45.1 or CD45.2 genotype can be performed using flow cytometry of bone marrow and peripheral blood as mentioned in section 7 in this protocol.
4. Mice after irradiation have compromised immunity. Keep mice in pathogen free facility.
5. Operation of irradiator requires special security clearance and training. To save time in training and approval processes, it is best to collaborate with a laboratory with qualified technician who is able to operate the irradiator.
6. Many institutions have flow cytometry laboratories with experienced technicians. To minimize problems with flow cytometry operation, it is better to discuss your flow cytometry experiment plan with the experienced technician before you start.
7. Mice were assigned in this manner (10 mice per group):

Group		Bone marrow donor to recipient	Treatment
A	BM exchange	CD45.1 WT to CD45.2 KO	DSS colitis
B			Water normal control
C	BM exchange	CD45.2 KO to CD45.1 WT	DSS colitis
D			Water normal control
E	Sham	CD45.1 WT to CD45.1 WT	DSS colitis
F			Water normal control
G	Sham	CD45.2 KO to CD45.2 KO	DSS colitis
H			Water normal control

* A brief illustration of experimental protocol is shown in Figure 1.

2. Recipient Mice Irradiation

1. Mice are bred and kept in pathogen-free facility. Place mice in autoclaved irradiation pie with filter. Place one mouse in each slot of irradiation pie. Place the pie into the irradiator and make sure the turntable and pie are turning.
2. Turn on the air-pump for ventilation. Close the irradiator door and lock it.
3. Irradiate the mice with 1,000 rad (which is equivalent to 10 Gy) around 10 min (depending on radiation source and half-life). From this time point, the irradiated mice are immuno-compromised and have weak immunity against infection. After irradiation, take the pie out and place in a sterile container for transportation to biosafety cabinet in animal facility. Avoid exposing the mice to external environment to minimize chance of infection.
4. In biosafety cabinet, transfer the mice into autoclaved mouse cages with autoclaved bedding (4 mice per cage. Add new sterile water with sulfatrim suspension - 3.12 ml per 100 ml water).
5. Wrap the water bottle with aluminum foil as the antibiotics are light sensitive. Shake to mix the sulfatrim solution bottle well before loading it to the cage.

3. Donor Bone Marrow Extraction

1. Sacrifice mice with carbon dioxide or isoflurance and then immerse in 1:200 Lysol solution. Spray the mice with 70% ethanol to wet the skin and dissect open bones.
2. Soak the dissection instruments in 70% ethanol for sterilization.
3. Perform the dissection in a biosafety cabinet. Use the sterile dissection instruments to expose the humerus, femur, tibia and fibula bones, free from attaching muscles and ligaments.
4. Cut open both ends of bones to show the red bone marrow. Hold the bones with forceps. Flush the red bone marrow out with cold PBS with 3 ml syringe and 25G needles to a Petri dish. Flush from both ends of bones to yield more bone marrow cells.
5. Use 6 ml syringes and 18G1/2 needles to break red bone marrow plugs by repeated aspiration and ejection. Transfer bone marrow with 50 ml Falcon tubes.
6. Spin the bone marrow down in 1,800 rpm for 5 min at 4 °C. Discard supernatant, resuspend pellet by vortexing for 5-10 sec.
7. Dilute 10X RBC lysis buffer with sterile water. Add 1X RBC lysis buffer (5 ml per donor mouse), vortex again for 5 sec and keep exactly 3 min at room temperature.
8. After 3 min, fill the tubes with 20 ml cold PBS to stop the lysis and mix. Pour the content through a 40 μm cell strainer directly into a new 50 ml Falcon tube.
9. Rinse the original tubes with 5 ml of PBS and transfer to cell strainer as well. Top up to 50 ml with PBS and mix.

4. Counting Bone Marrow Donor Cells

1. Add 100 μl of Trypan blue to an Eppendorf tube and 100 μl of bone marrow suspension to an Eppendorf tube and mix. Pipette the stained cell mixture to a hemocytometer.
2. The amount of cells is calculated by total living bone marrow cell count in the Falcon tubes = unstained cell number in 16 squares x 2 (due to Trypan blue dilution) x 10,000 x 50 ml
3. Spin down the bone marrow cells in 50 ml Falcon tubes at 2,000 rpm for 5 min at 4 °C.
4. Remove the supernatant. Based on the total cell counts, resuspend the pellet with PBS to 1 x 10⁸ cells per ml

5. Infusion to Recipient Mice

1. Warm the recipient mice on a heat pad and under a warming lamp. Put the recipient mice in restrainer. Inject 1 x 10⁷ cells per mouse in 100-200 μl intravenously.
2. Injection of donor bone marrow must be done between 4-24 hr after irradiation.
3. Keep up to 4 injected mice per cage. Maintain the injected recipient mice in autoclaved cages with antibiotics-treated water for the first 4 weeks in pathogen free animal facility and let the mice regain immunity. Change cages, antibiotic-treated water and food every 4 days to maintain hygiene.

6. Induction of Colitis and Evaluation of Colitis

1. 4 weeks after irradiation and bone marrow transplantation, switch to regular water without antibiotics and maintain the mice for 2 more weeks to regain their normal gut microflora.
2. 6 weeks after bone marrow injection, measure initial body weight. Some mice are given 5% dextran sulfate in drinking water ad libitum to induce colitis. Some mice are given regular drinking water only as normal control groups.
3. 5 days later, withdraw peripheral blood, transfer to heparin coated tubes (Vacutainer) and keep in ice. Pool the blood from 4 mice to 1 tube. Combine 300 μl of blood with 300 μl of cell staining buffer = 600 μl. Divide the blood to 5 Eppendorf tubes, each with 100 μl.
4. Sacrifice the mice with carbon dioxide or isoflurane. Evaluate macroscopic damage scores (please refer to another JOVE video publication for details ¹). Measure body weight change. Measure bowel length and thickness with digital caliper. Observe stool texture (hard, soft or bloody). Determine occult blood in stool with Hemooccult. Dissect colon tissues and fix in formalin for H&E staining.
5. Dissect 1 femur bone per mouse, extract bone marrow and flush the bone marrow with cold PBS via 25G needle and 1 ml syringe. Pool 4 bone marrow plugs to 1 tube and keep in ice.
6. Pour the bone marrow to Petri dish, shear the bone marrow plug with 18G needle and 5 ml syringe several times until no bone marrow plug is present.
7. Spin down with 1,500 rpm for 5 min at 4 °C. Remove supernatant and resuspend with 600 μl cell staining buffer. Divide the resuspended bone marrow to 5 Eppendorf tubes, each with 100 μl.

7. Quality Inspection of Bone Marrow Transplantation by Flow Cytometry

1. Label each sample tubes of blood or bone marrow #1-5:
2. Prepare the antibody mixture for each respective tube in the dark.

#1 No antibody
#2 30 μl FITC isotype control + 30 μl PE isotype control + 15 μl CD16/32 blocking
#3 30 μl FITC CD45.1 Ab + 15 μl CD16/32 blocking
#4 30 μl PE CD45.2 Ab + 15 μl CD16/32 blocking
#5 30 μl FITC CD45.1 Ab + 30 μl PE CD45.2 Ab + 15 μl CD16/32 blocking

Add nothing to blood or bone marrow sample #1

Add 5 μl of antibody mixture #2 to each blood or bone marrow sample #2

Add 3 μl of antibody mixture #3 to each blood or bone marrow sample #3

Add 3 μl of antibody mixture #4 to each blood or bone marrow sample #4

Add 5 μl of antibody mixture #5 to each blood or bone marrow sample #5

3. Keep in dark in ice for 30 min.
4. Add 2 ml 1X RBC lysis buffer to each tube. Keep in dark on ice for 15 min.
5. Spin down the cells at 1,500 rpm, 5 min at 4 °C. (GH 3.8 rotor, 1,500 rpm = 350 x g) Remove supernatant. Resuspend the pellet with 500 μl cell staining buffer in the dark, then vortex briefly.
6. Check the CD45.1 (representing WT) and CD45.2 (representing KO) in the immunostained blood and bone marrow samples by flow cytometry. Select FITC to represent WT CD45.1 and PE to represent KO CD45.2.
7. Analyze the flow cytometry results by FlowJo software. Calculate the ratio of FITC:PE ratio or PE:FITC ratio to determine whether donor genotype is dominant genotype in blood and bone marrow.

Results

If the gene-of-interest plays a significant role in immune cells during development of colitis, the mice receiving bone marrow of different genotype through bone marrow transplantation (WT to KO or KO to WT) should have an altered response to DSS colitis. One of the most important parameters for determining the severity of colitis is the H&E staining of the colonic tissues. Colonic tissue structure changes and signs of inflammation can be quantitatively evaluated by H&E histology scoring system. Criteria for H&am...

Discussion

This bone marrow transplantation approach is suitable for immunology research of colitis, infection, cancer, obesity and other diseases. This bone marrow transplantation experiment is needed when the gene-of-interest is expressed in both bone marrow derived and non-bone marrow derived cells and the gene-of-interest is suspected to mediate disease by cells from either population. For example, antimicrobial peptide cathelicidin is shown to modulate acute colitis. But it is expressed in both epithelial cells and immune cell...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell staining buffer	Biolegend	#420201
10X RBC lysis buffer	Biolegend	#420301
FITC mouse isotype control	Biolegend	#400207
PE mouse isotype control	Biolegend	#400211
PE anti-mouse CD45.2	Biolegend	#109807
FITC anti-mouse CD45.1	Biolegend	#110705
Anti-mouse CD16/32 blocking	Biolegend	#101301
40 μm cell strainer	Fisherbrand	#22363547
PBS 1X	MP biomedicals	#1860454
Heparin	Fisherbrand	#BP2425
Sulfatrim (SMZ)	Qualitest	NC9242720 (fisher)
Lysol IC	Andwin Scientific	NC9745686 (fisher)
Vaccutainer	BD	#8000813
Mouse restrainer	Braintree Scientific	#TV-150
Irradiator	J.L. Shepherd and Associates	Mark I 68A
Flow cytometer	BD	BD FACSCanto II
Flow cytometer test-tube	Falcon	#352052
Digital caliper	Fisherbrand	14-648-17
Hemoccult ICT	Beckman Coulter	G0328QW