Techniques for Modeling Human Intestinal Cancer in Mice

October 22-26, 2000

Presented by

The Jackson Laboratory

Faculty: Catherine Booth, William F. Dove, Jeffrey I. Gordon, Richard B.
Halberg, Julie Ann O\'Shea, Thaddeus S. Stappenbeck, and Robert H.
Whitehead

Edited by: W. F. Dove, R. B. Halberg, L. Clipson,

and I. L. Riegel

The McArdle Laboratory for Cancer Research

University of Wisconsin-Madison

Send any corrections or additions to dove@oncology.wisc.edu

6\. **Tumor Scoring and Other Biological Protocols**

# Isolation and Analysis of the Intestinal Tract and Its Lesions

# Dissecting

# Notes

# Supplies

# Scoring Intestinal Adenomas

# Examples

# Isolating and Embedding Intestinal Adenomas 

# Supplies

i.  **Staining Sections with H&E**

ii. **Solutions**

iii. **Examples**

```{=html}
<!-- -->
```
a.  **Detecting β-galactosidase Activity in Whole Mounts**

    i.  **Preparation and Staining Tissue**

    ii. **Notes**

    iii. **Solutions**

    iv. **Examples**

b.  **Immunohistochemistry**

    i.  **Antigen Presentation**

    ii. **Antigen Detection**

    iii. **Staining with DAB**

    iv. **Staining with Fluorescently Labeled Antibodies**

    v.  **Notes**

    vi. **Solutions**

    vii. **Antibodies**

    viii. **Supplies**

    ix. **Examples**

c.  **Quantitative PCR Assay to Monitor Status of *Apc*^+^**

    i.  **Preparation of DNA from Sections**

    ii. **Assay**

    iii. **Solutions**

    iv. **Example**

d.  **Generation of Aggregation Chimeras**

    i.  **Overview**

    ii. **Detailed Procedure**

    iii. **Notes**

    iv. **Supplies**

    v.  **Examples**

# Isolation and Analysis of the Intestinal Tract and Its Lesions

**RB Halberg and WF Dove**

**McArdle Laboratory, University of Wisconsin**

# 

# [Dissecting]{.underline}

# 

# Sacrifice mouse by CO~2~ asphyxiation.

# Wet fur in the abdominal region with 70% ethanol.

1.  Make an incision in the skin that transverse the abdomen.

2.  Pull skin so that the body wall is exposed from forelimbs to hind
    limbs.

3.  Make an incision in the body wall that transverses the abdomen.

4.  Remove the intestinal tract.

    a.  Cut the colon as near to the rectum as possible.

    b.  Grasp the colon with the dissection forceps.

    c.  Pull gently to release colon.

    d.  Grasp mesentery that is attached to the small intestine near the
        cecum.

    e.  Continue to pull to begin stripping off the mesentery.

    f.  Grasp small intestine with dissection forceps.

    g.  Continue to pull to release the small intestine.

    h.  Repeat in a hand-over-hand fashion up to the stomach.

    i.  Cut the stomach at the junction with the small intestine.

    j.  Place intestine in PBS.

5.  Cut small intestine at the junction between with the cecum.

6.  Flush small intestine with PBS.

    a.  Attach a reusable feeding needle to a 10 ml syringe.

    b.  Draw up PBS.

    c.  Insert needle in the small intestine.

    d.  Wash out contents of the intestinal lumen by injecting PBS.

7.  Cut small intestine into four equal segments.

    a.  Loop small intestine over a pair of dissection forceps so that
        the ends of meet.

    b.  Hold tissue just below the halfway point with a second pair of
        dissection forceps.

    c.  Cut the small intestine at the halfway point.

    d.  Loop two segments over a pair of dissection forceps so that the
        ends meet.

    e.  Hold tissue just below the halfway point with a second pair of
        dissection forceps.

    f.  Cut the two segments at the halfway point.

    g.  Lay four segments on a paper towel and trim off any remaining
        mesentery.

8.  Rinse segments in PBS and lay them onto on a piece of bibulous
    paper.

9.  Cut longitudinally the entire length of each segment by using
    microdissecting scissors with probed points. Avoid cutting tumors in
    half, if possible.

10. Dip finger in PBS and run the length of each segment so that the
    intestine is flattened out. Villi and tumors should be exposed.

11. Cut colon at the junction with the cecum.

12. Wash the colon in PBS and place it on bibulous paper.

13. Cut longitudinally the entire length of the colon.

14. Remove fecal pellets with dissection forceps.

15. Dip finger in PBS and run the length of the colon to flatten it out.

16. Wash segments and colon with 70% ethanol while running finger up and
    down the length of each segment and colon.

17. Cut bibulous paper around two of the four segments of the small
    intestine.

18. Fold in half or thirds so that the sample will fit into the
    scintillation vial containing 10% formalin.

19. Repeat for the remaining two segments of the small intestine and the
    colon.

20. Fix tissue overnight.

21. Replace 10% formalin with 70% ethanol.

22. Tissue can be stored in this manner for years. Long-term storage
    requires sealing the cap of the scintillation vial with parafilm.

## [Notes]{.underline}

1.  The intestinal tract can tear quite easily, especially in mice that
    have several tumors.

2.  Tumors in the colon can be dislodged very easily as they are
    attached to the mucosa by a stem-like structure.

## [Supplies]{.underline}

+-----------------------------------+-------+-------+---------+-------+
| #### Item                         | ##    | S     | Catalog | ####  |
|                                   | ### A | ource | No.     | Price |
+-----------------------------------+-------+-------+---------+-------+
|                                   |       |       |         |       |
+-----------------------------------+-------+-------+---------+-------+
| Forceps, microdissecting          | 2     | Roboz | RS-5130 | \$    |
|                                   |       |       |         | 26.95 |
+-----------------------------------+-------+-------+---------+-------+
| Scissors, veterinary              | 1     | Roboz | RS-6940 | \$    |
|                                   |       |       |         | 73.61 |
+-----------------------------------+-------+-------+---------+-------+
| Scissors, microdissecting         | 1     | Roboz | RS-5910 | \$    |
|                                   |       |       |         | 43.27 |
+-----------------------------------+-------+-------+---------+-------+
| Scissors, microdissecting with    | 1     | Roboz | RS-5990 | \$    |
| probed points                     |       |       |         | 67.30 |
+-----------------------------------+-------+-------+---------+-------+
| Needle, reusable feeding          | 1     | Roboz | FN-7922 | \$    |
|                                   |       |       |         | 19.40 |
+-----------------------------------+-------+-------+---------+-------+
| Bibulous paper, (15 cm x 10 cm),  |       | F     | 11998   | \$    |
| pk of 12                          |       | isher |         | 21.42 |
+-----------------------------------+-------+-------+---------+-------+
| Syringe, 10 ml, pk of 100         |       | F     | 14      | \$    |
|                                   |       | isher | -823-2A | 20.15 |
+-----------------------------------+-------+-------+---------+-------+
| Petri dish, (100 mm x 15 mm),     |       | F     | 08      | \$    |
| case of 500                       |       | isher | -757-12 | 93.60 |
+-----------------------------------+-------+-------+---------+-------+
| Scintillation vials, case of 500  |       | F     | 0       | \$1   |
|                                   |       | isher | 3-337-4 | 36.50 |
+-----------------------------------+-------+-------+---------+-------+
| Wash bottle, pk of 4              |       | F     | 03-     | \$    |
|                                   |       | isher | 409-10D | 14.11 |
+-----------------------------------+-------+-------+---------+-------+
| Paper toweling                    |       |       |         |       |
+-----------------------------------+-------+-------+---------+-------+

## [Scoring Intestinal Adenomas]{.underline}

1.  Remove intestinal tract from the scintillation vial.

2.  Remove each segment of the small intestine and colon from the
    bibulous paper and place them in a large petri dish containing 70%
    ethanol.

3.  Trim off any mesentery that may interfere with scoring intestinal
    tumors.

4.  Score the number of intestinal tumors in each segment of the small
    intestine and the colon under low magnification. Difficulties
    include confusing either the ampulla of Vader, cystic crypts, or
    lymph nodes as tumors and discriminating between tumors that are
    tightly clustered.

[Examples]{.underline}

+-----------------------------------+-----------------------------------+
| ![](media/image1.png)A. Adenomas  | ![](media/image3.png)             |
| (a), cystic crypt (c) and lymph   |                                   |
| node                              | B. Small adenoma (a) and cystic   |
| (n)![](media/image2.w             | crypt (c)                         |
| mf){width="0.12013888888888889in" |                                   |
| height="0.12430555555555556in"}   |                                   |
+-----------------------------------+-----------------------------------+
| ![](media/image4.png)             | ![](me                            |
|                                   | dia/image5.png)![](media/image2.w |
| C. Flat adenoma (a) and adenoma   | mf){width="0.12013888888888889in" |
| cluster (aa)                      | height="0.12430555555555556in"}   |
|                                   |                                   |
|                                   | D. Ampulla of Vader (v)           |
+-----------------------------------+-----------------------------------+
| ![](media/image6.png)             | ![](media/image7.png)             |
|                                   |                                   |
| E. Lymph node (n) and adenoma (a) | F. Adenoma cluster (aa) in colon  |
| in colon                          |                                   |
+-----------------------------------+-----------------------------------+

[Isolating and Embedding Intestinal Adenomas]{.underline}

1.  Individual tumors can be isolated for histological examination with
    microdissecting scissors.

# 

# 

# 

# 

# The biopsy is embedded in paraffin.

![](media/image8.png){width="3.1902777777777778in"
height="1.9534722222222223in"}

# 

# 

# 

# 

# 

# 

# 

# 

2.  Manipulations may vary with the experiment. We have found it
    convenient to embed several tumors from an individual mouse in one
    block. Tumors are arranged in a pattern to facilitate
    identification.

# 

# 

# [Supplies]{.underline}

  -------------------------- ------------ -------- -------------- ----------
  Item                       Amount       Source   Catalog No.    Price

                                                                  

  Tweezers, Dumont #5        2            Roboz    RS-5045        \$20.74

  Scissors, Microdissecting  1            Roboz    RS-5620        \$239.00

  15 mm x 150 mm petri dish  Case of 100  Fisher   08-757-14      \$95.00
  -------------------------- ------------ -------- -------------- ----------

[Staining sections with hematoxylin and eosin]{.underline}

1.  Deparaffinize sections in three changes of xylene for 2 min each.

2.  30 dips in 2 changes of absolute alcohol.

3.  15 dips, then 30 sec in 80% ethanol.

4.  15 dips, then 30 sec in 70% ethanol.

5.  Distilled H~2~0 for 1 min. Repeat 4 times.

6.  Stain in Shandon Instant Hematoxylin for 30 sec.

7.  Wash in tap water for 3 min.

8.  3 dips in 1% alcoholic HCl.

9.  Rinse in tap water for 1 min.

10. Wash sections in 0.4% sodium acetate for 3 min.

11. 10 dips in H~2~0. Repeat three times.

12. Wash sections in 50% ethanol for 30 sec.

13. Stain in eosin-multichrome stain for 30 sec.

14. 30 dips in absolute ethanol. Repeat three times.

## [Solutions]{.underline}

#### *Shandon Instant Hematoxylin*

Mix parts A and B dry concentrate in 1 L of distilled H~2~O.

Wait at least 12 hr and filter.

***1% alcoholic HCl***

10 ml Concentrated HCl

300 ml Distilled H~2~O

700 ml Absolute ethanol

Add HCl to distilled H~2~O, then absolute ethanol.

***0.4% sodium acetate***

1 g sodium acetate

250 ml distilled H~2~O

Mix well and adjust pH to 7.25.

Make fresh.

#### *eosin-multichrome stain*

2.5 g Eosin Y

0.25 g Phloxine B

500 ml Distilled H~2~O

500 ml Absolute ethanol

2 ml Glacial acetic acid

Dissolve eosin and phloxine B in distilled H~2~0. Add ethanol, then
glacial acetic acid. pH must be 5.

[Examples]{.underline}

+-----------------------------------------------------------------------+
| ![](media/image9.png)                                                 |
|                                                                       |
| A. Small intestine tumor, H&E                                         |
+-----------------------------------------------------------------------+
| ![](media/image10.png)                                                |
|                                                                       |
| B. Colon tumor, H&E                                                   |
+-----------------------------------------------------------------------+

**Detecting β-galactosidase Activity in Whole Mounts**

**RB Halberg, A Thliveris and WF Dove**

**McArdle Laboratory, University of Wisconsin**

## [Preparation and Staining of Tissue]{.underline}

1.  Remove intestinal tract and flush it with PBS.

2.  Lay intestine out on bibulous paper.

3.  Pin tissue and paper to paraffin wax.

4.  Fix tissue in 4% paraformaldehyde for 1 hr at 4ºC.

5.  Wash tissue in rinse buffer for 30 min at room temperature. Repeat
    two times.

6.  Stain overnight at 37ºC.

7.  Fix tissue in 10% formalin for at least 12 hr at room temperature.

8.  Wash and store in 70% ethanol.

## [Notes]{.underline}

1.  Do not wash tissue with ethanol when dissecting.

2.  We pour paraffin into a 25 mm x 150 mm plate that has been scratched
    extensively and allow it to cool at room temperature.

3.  Other fixatives such as glutaraldehyde can be used, but we have had
    the best results with 4% paraformaldehyde.

4.  Fixation must be done at 4ºC.

5.  Xgal is light sensitive. We weigh out enough for each experiment and
    then cover it with nitrogen and store in the dark at --20ºC.

6.  Xgal can be dissolved in DMF or DMSO. We use DMF.

## [Solutions]{.underline}

***0.1 M Sodium phosphate buffer***

115 ml 0.1 M sodium phosphate, monobasic

385 ml 0.1 M sodium phosphate, dibasic

***4% Paraformaldehye***

20 g paraformaldehyde

100 ml PBS

Heat 65ºC with stirring for at least hours.

Freeze 20ml aliquots.

Thaw an aliquot for each experiment.

Add 80 ml PBS.

#### *Rinse buffer*

0.4 ml 1 M MgCl~2~

2.0 ml 1% sodium deoxycholate

2.0 ml 2% IGEPAL

196 ml 0.1 M sodium phosphate

buffer

#### *Stain*

50 mg Xgal

2 ml DMF

0.106 g potassium ferrocyanide

0.082 g potassium ferricyanide

48 ml rinse buffer

Dissolve Xgal in DMF and then add to rinse buffer.

# [Example]{.underline}

# 

+-----------------------------------+-----------------------------------+
| ![](media/image11.png)            |                                   |
|                                   |                                   |
| A. Small intestine from chimera   |                                   |
| stained with Xgal                 |                                   |
+-----------------------------------+-----------------------------------+
| ![](media/image12.png)            | ![](media/image13.png)            |
|                                   |                                   |
| B. Section of a polyclonal tumor  | C. Enlargement of B.              |
| from a chimera                    |                                   |
+-----------------------------------+-----------------------------------+

# Immunohistochemistry

**RB Halberg and WF Dove**

**McArdle Laboratory, University of Wisconsin**

## [Antigen Presentation]{.underline}

1.  Deparaffinize for 1 hr in xylene.

2.  Incubate for 10 min in 100% ethanol.

3.  Rehydrate through ethanol series: 90, 80, and 70%. Incubate for 5
    min in each solution.

4.  Microwave for 25 min on high power in citrate buffer.

5.  Cool in running water.

6.  Wash for 5 min in double-distilled water.

7.  Wash for 10 min in PBS.

## [Antigen Detection]{.underline}

1.  Wipe out area around sections with a Wexal-stick. DO NOT ALLOW
    SECTIONS TO DRY OUT.

2.  Block endogenous peroxidase activity by incubating for 15 min in
    0.3% H~2~O~2~/PBS at room temperature.

3.  Wash for 10 min in PBS.

4.  Block in 10% native goat serum/PBS. Incubate for 30 min in humidity
    chamber at room temperature.

5.  Tap off serum and add primary antibody to one section and PBS to the
    other section as a negative control. Incubate for 1 hr at room
    temperature or overnight at 4°C.

6.  Wash in PBS for 5 min. Repeat 2 times.

7.  Wash in Tween20/PBS (100 μl/600 ml) for 1 hr.

[Staining with DAB]{.underline}

1.  Add a 1:200 dilution of biotinylated goat anti-rabbit IgG (H+L) in
    PBS to both sections. Incubate for 1 hr at room temperature.

2.  Make up ABC reagent. Add 50 μl of reagent A to 2.5 ml of PBS. Mix.
    Add 50 μl of Reagent B. Mix

3.  Wash for 5 min in PBS. Repeat two times.

4.  Add ABC to all sections for 30 min at room temperature.

5.  Wash for 5 min in PBS. Repeat 2 times.

6.  Develop in DAB solution for 5 min in the dark.

7.  Check for staining and then wash under tap water for 5 min.

8.  Wash in double-distilled water for 10 min.

9.  Counterstain with 1:3 dilution of hematoxylin/H20. Usually, 10
    seconds is long enough.

10. Wash under tap water until no color is lost.

11. Dehydrate through ethanol series: 70, 80, 90%. Incubate for 5 min in
    each solution.

12. Incubate for 10 min in 100% ethanol.

13. Incubate for 30 min in xylene.

14. Coverslip

## [Staining with Fluorescently Labeled Antibodies (Alternative protocol)]{.underline}

1.  Add a 1:100 dilution of FITC-labeled goat anti-rabbit IgG in PBS to
    both sections. Incubate for 1 hr at 37°C.

2.  Wash for 5 min in PBS. Repeat.

3.  Wash for 5 min in DAPI/PBS (1:1000 dilution of 1mg/ml DAPI stock)

4.  Add one drop of antifade.

5.  Coverslip

6.  Seal with fingernail polish.

7.  Store at 4°C.

### Notes

1.  Make sure that you use citric acid and not sodium citrate when
    making the citrate buffer.

2.  When using cotton swabs to dry area around the section, make sure
    strings from the cotton swab do not disrupt the section.

3.  H~2~O~2~ stocks are prepared as a 30% solution and stored in the
    refrigerator. Keep this in mind when calculating the dilution.

4.  Some of the primary antibodies are not commercially available, so
    use sparingly.

5.  The primary and secondary antibodies can be diluted in PBS or 0.5%
    nonfat dry milk/ PBS solution. The latter reduces nonspecific
    interactions and thereby background staining.

6.  ABC reagent is very expensive. Make only what you need.

7.  The DAB solution contains H~2~0~2~. This component is a necessary
    catalyst.

8.  When developing the slides in DAB solution, be careful not to
    overstain. This step should be constantly monitored and never takes
    longer than a couple of minutes.

[Solutions]{.underline}

#### *Citrate buffer*

2.1 grams citric acid

1 gram sodium hydroxide

H~2~0 to 1 L

#### *DAB solution*

0.25 mg/ml DAB

0.03% H~2~O~2~

200 ml PBS

### 

### pH will be approximately 6.

### 

### 

### Antibodies

[Supplies]{.underline}

  ---------------- ----------- ----------------- --------------- ----------
  Item             Amount      Source            Catalog No.     Cost

                                                                 

  Wexal-stick      Box of 144  Walla Medical     S-0008680       \$29.60

  Humidity chamber             Shandon Lipshaw   197             \$145.00

  Native goat                  Vector            S-1000          \$35.00
  serum                        Laboratories                      

  ABC reagent      Kit         Vector            PK-6100         \$155.00
                               Laboratories                      

  DAB              5 g         Sigma             D5367           \$39.70

  Antifade                     Oncor             S-1331-2        
  ---------------- ----------- ----------------- --------------- ----------

[Examples]{.underline}

+-----------------------------------+-----------------------------------+
| D5877B                            | D5877J                            |
|                                   |                                   |
| ![](media/image14.png)            | ![](media/image15.png)            |
|                                   |                                   |
| A. H&E                            | B. H&E                            |
+-----------------------------------+-----------------------------------+
| ![](media/image16.png)            | ![](media/image17.png)            |
|                                   |                                   |
| C. 3122 (green) and DAPI (blue)   | D. 3122 (green) and DAPI (blue)   |
+-----------------------------------+-----------------------------------+
|                                   | ![](media/image18.png)            |
|                                   |                                   |
|                                   | E. Negative control for 3122      |
+-----------------------------------+-----------------------------------+

# Quantitative PCR Assay to Monitor the Status of *Apc^+^*

# 

# [Preparation of DNA from Sections]{.underline}

# 

# Scrape tumor from 5--10 slides with a scalpel. Use as a guide a serial section that has been stained with H&E.

1.  Suspend sample in 20 μl of digestion buffer.

2.  Incubate overnight at 37ºC.

3.  Add 3 μl of Chelex slurry to each sample.

4.  Vortex lightly.

5.  Incubate for 1 hr at 37ºC.

6.  Boil samples for 5 min.

7.  Spin at high speed for 5 min.

8.  Store at -20ºC.

## [Assay]{.underline}

1.  Prepare master mix.

> Component (per reaction) Amount

10 x PCR buffer 2.0 μl

25 mM MgCl~2~ 1.6 μl

10 mM dNTP 0.4 μl

20 μM MAPC H3 F 0.4 μl

20 μM MAPC H3 R 0.4 μl

Vortex.

Taq polymerase 0.4 μl

3.3 μM dCTP^32^ 0.2 μl

Invert to mix.

2.  Add 4.6 μl of sample.

3.  Run program.

  --------------- ----------------------- -------------------------------
                                          

  Step 1          94ºC                    3 min

  Step 2          94ºC                    15 sec

  Step 3          60ºC                    2 min

  Step 4          72ºC                    2 min

  Step 5          29 times to 2           

  Step 6          72ºC                    7 min

  Step 7          15ºC                    1 min

  Step 8          4ºC                     
  --------------- ----------------------- -------------------------------

4.  Add 5 μl of *Hin*dIII mix.

> Component (per reaction) Amount

50 mM Spermidine 1.5 μl

*Hin*dIII Buffer 1.5 μl

Distilled H~2~O 0.5 μl

*Hin*dIII 1.5 μl

5.  Incubate overnight at 37ºC.

6.  Add 3 μl 6 x loading buffer.

7.  Load samples on the gel. We use a 7.5% polyacrylamide gel with TBE
    as the running buffer.

8.  Run at 150 V until dye front reaches the bottom of the gel.

9.  Transfer gel to 3MM Whatman filter paper and dry with heat for 1 hr.

10. Measure signals with a phosphoimager.

11. Calculate ratio of wildtype allele to Min allele. The product from
    the Min allele is longer and contains more C nucleotides, so the
    value has to be multiplied by a factor of 0.85.

## [Notes]{.underline}

1.  The chelex slurry will interfere with the PCR assay, so avoid it
    when taking the sample for analysis.

2.  Each sample is assayed at least twice. If the ratios obtained vary
    by more than 20%, then the sample is discarded.

3.  A value of less than 0.3 indicates the wildtype allele has been
    lost; a value greater than 0.6 indicates the wildtype allele has
    been maintained.

[Solutions]{.underline}

***Digestion buffer***

1% Tween 20

0.2 mM EDTA

20 mM Tris, pH 8

Combine equal volumes of this buffer and a 2 mg/ml proteinase K stock.

#### 

#### *Chelex slurry*

Chelex resin

0.1 mM EDTA

10 mM Tris, pH 8

The resin does not go into solution.

## [Primers]{.underline}

# 

# MAPC H3 F 5' TCTCGTTCTGAGAAAGACAGAAGCT 3'

# MAPC H3 R 5' TGATACTTCTTCCAAAGCTTTGGCTAT 3'

# 

# [Example]{.underline}

# 

# ![](media/image19.png)

# 

+------+--------+-------+------+---------------+---------------+------+
| #    | **     | **S   | **Bk | **Signal-Min  | **Signal-Wt   | *    |
| #### | Signal | ignal | gd** | (corrected)** | (corrected)** | *Rat |
| # Sa | -Min** | -Wt** |      |               |               | io** |
| mple |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
|      |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
| T    | 3      | 6     | 3    | 3030348.15    | 619306        | 0.20 |
| umor | 601239 | 50011 | 0705 |               |               | 4368 |
| 1    |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
| No   | 1      | 7     | 2    | 1060408.1     | 764371        | 0.72 |
| rmal | 282426 | 94025 | 9654 |               |               | 0827 |
| 1    |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
|      |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
| T    | 2      | 5     | 2    | 2026348.9     | 536514        | 0.26 |
| umor | 415474 | 63318 | 6804 |               |               | 4769 |
| 2    |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
| No   | 5      | 29    | 2    | 4416921.95    | 2879841       | 0.65 |
| rmal | 230367 | 08731 | 8890 |               |               | 2002 |
| 2    |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
|      |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
| T    | 908016 | 3     | 2    | 742923.6      | 279856        | 0.37 |
| umor |        | 08746 | 8890 |               |               | 6696 |
| 3    |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
| No   | 821227 | 5     | 3    | 661529.95     | 552204        | 0.83 |
| rmal |        | 88717 | 6513 |               |               | 4738 |
| 3    |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
|      |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
| T    | 6      | 20    | 3    | 5126392.5     | 1989516       | 0.38 |
| umor | 073530 | 25624 | 6108 |               |               | 8093 |
| 4    |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
| No   | 5      | 44    | 3    | 4505072.05    | 4392497       | 0.97 |
| rmal | 344853 | 30550 | 8053 |               |               | 5011 |
| 4    |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
|      |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
| Note |        |       |      |               |               |      |
| that |        |       |      |               |               |      |
| the  |        |       |      |               |               |      |
| c    |        |       |      |               |               |      |
| orre |        |       |      |               |               |      |
| cted |        |       |      |               |               |      |
| si   |        |       |      |               |               |      |
| gnal |        |       |      |               |               |      |
| for  |        |       |      |               |               |      |
| the  |        |       |      |               |               |      |
| Min  |        |       |      |               |               |      |
| al   |        |       |      |               |               |      |
| lele |        |       |      |               |               |      |
| eq   |        |       |      |               |               |      |
| uals |        |       |      |               |               |      |
| the  |        |       |      |               |               |      |
| raw  |        |       |      |               |               |      |
| si   |        |       |      |               |               |      |
| gnal |        |       |      |               |               |      |
| m    |        |       |      |               |               |      |
| inus |        |       |      |               |               |      |
| the  |        |       |      |               |               |      |
| ba   |        |       |      |               |               |      |
| ckgr |        |       |      |               |               |      |
| ound |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
| and  |        |       |      |               |               |      |
| then |        |       |      |               |               |      |
| mu   |        |       |      |               |               |      |
| ltip |        |       |      |               |               |      |
| lied |        |       |      |               |               |      |
| by a |        |       |      |               |               |      |
| fa   |        |       |      |               |               |      |
| ctor |        |       |      |               |               |      |
| of   |        |       |      |               |               |      |
| 0    |        |       |      |               |               |      |
| .85, |        |       |      |               |               |      |
| whe  |        |       |      |               |               |      |
| reas |        |       |      |               |               |      |
| the  |        |       |      |               |               |      |
| c    |        |       |      |               |               |      |
| orre |        |       |      |               |               |      |
| cted |        |       |      |               |               |      |
| si   |        |       |      |               |               |      |
| gnal |        |       |      |               |               |      |
| for  |        |       |      |               |               |      |
| the  |        |       |      |               |               |      |
| wild |        |       |      |               |               |      |
| type |        |       |      |               |               |      |
| al   |        |       |      |               |               |      |
| lele |        |       |      |               |               |      |
| is   |        |       |      |               |               |      |
| the  |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
| raw  |        |       |      |               |               |      |
| si   |        |       |      |               |               |      |
| gnal |        |       |      |               |               |      |
| m    |        |       |      |               |               |      |
| inus |        |       |      |               |               |      |
| the  |        |       |      |               |               |      |
| bac  |        |       |      |               |               |      |
| kgro |        |       |      |               |               |      |
| und. |        |       |      |               |               |      |
| The  |        |       |      |               |               |      |
| fa   |        |       |      |               |               |      |
| ctor |        |       |      |               |               |      |
| of   |        |       |      |               |               |      |
| 0.85 |        |       |      |               |               |      |
| corr |        |       |      |               |               |      |
| ects |        |       |      |               |               |      |
| for  |        |       |      |               |               |      |
| the  |        |       |      |               |               |      |
| nu   |        |       |      |               |               |      |
| mber |        |       |      |               |               |      |
| of C |        |       |      |               |               |      |
| nuc  |        |       |      |               |               |      |
| leot |        |       |      |               |               |      |
| ides |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+
| in   |        |       |      |               |               |      |
| each |        |       |      |               |               |      |
| prod |        |       |      |               |               |      |
| uct. |        |       |      |               |               |      |
+------+--------+-------+------+---------------+---------------+------+

#  Generation of Aggregation Chimeras

**RB Halberg, A Thliveris and WF Dove**

**McArdle Laboratory, University of Wisconsin**

## [Overview]{.underline}

## Day 1

-   Inject female donors with Pregnant Mare Serum (PMS).

Day 2

-   Wait.

Day 3

-   Inject female donors with Human Chorionic Gonadotrophin (HCG).

-   Set up female donors with males.

Day 4

-   Pull female donors that were plugged.

-   Set up ICR females with vasectomized ICR males.

Day 5

-   Pull ICR females that were plugged.

-   Prepare media and plates.

Day 6

-   Prepare other plates.

-   Isolate embryos from female donors and generate aggregation
    chimeras.

Day 7

-   Implant aggregation chimeras into ICR females.

Day 24-26

-   Aggregation chimeras will be born and fostered.

[Detailed Procedure]{.underline}

Day 1

1.  Inject female donors with PMS at 5:00PM. This hormone is available
    from Sigma in two different quantities: 50 I.U. or 1000 I.U. We
    purchase the latter and prepare a 50 I.U./ml stock in which the
    hormone is suspended in 0.9% NaCl. This stock is dispensed in 1-ml
    aliquots and is stable for 6 months when stored at -20°C.
    Alternatively, PEG 600 is available from Intervet. This source
    contains PMS and HCG. Some researchers contend that they get a
    greater yield of embryos with this combination of hormones, but we
    do not observe much difference between this combination and that
    available from Sigma.

Day 3

1.  Inject female donors with HCG at 5:00PM. This hormone is available
    from Sigma in two different quantities: 50 I.U or 10,000 I.U. We
    purchase the latter and prepare a 500 I.U./ml stock in which this
    hormone is suspended in H~2~0. This stock is dispensed into 100-μl
    aliquots and is stable for 6 months when stored at -20°C. We add 900
    μl of H~2~0 to the stock and inject the appropriate amount of
    hormone for the strain under investigation.

2.  Mate female donors with males.

Day 4

1.  Pull female donors that were plugged.

2.  ![](media/image20.png){width="2.6944444444444446in"
    height="1.5833333333333333in"}Set up ICR females with vasectomized
    ICR males. We set up 24-36 females with 12 males to ensure that we
    obtain enough pseudopregnant ICR recipients. Males must be
    vasectomized at least 10 days before the matings are set up and can
    be used for several months.

    a.  Anesthetize male.

    b.  Shave abdomen of the mouse.

    c.  Open the body wall by cutting skin with dissection scissors,
        making a 1.5 cm incision that is level with the top of the legs.

    d.  Pull the fat pad attached to the testis in order to remove
        tissue from the body cavity.

    e.  Hold vas deferens with a one pair of dissection forceps and
        cauterize with the red-hot tips of a second pair of forceps.

> ![](media/image21.png){width="3.129861111111111in"
> height="0.6069444444444444in"}

f.  Repeat steps for other testis.

g.  Slide tissue back into the body cavity.

h.  Close body wall with absorbent suture such as Vicryl.

i.  Close incision with wound clips.

Day 5

1.  Pull ICR females that were plugged.

2.  Prepare media.

    a.  M2, M16 and Tyrode's solution are available from Sigma. We tried
        alternative sources but found that embryos did not develop as
        well.

    b.  Add antibiotics and BSA to M2 and M16.

        i.  Prepare a 100x antibiotic stock, which consists of 10,000
            units/ml penicillin G and 5,000 μg/ml streptomycin sulfate.
            This stock is stored at --20°C and stable indefinitely.

        ii. Add 100 μl of this stock to 10 ml of each type of medium.
            Mix. Add 40 mg of BSA. Let stand for 10 min until BSA is in
            solution and then filter sterilize. The medium is stored at
            4°C and stable for 1 week.

3.  Prepare plates in which aggregation chimeras will be generated.

    a.  Make 12 wells in a 10 x 60 mm tissue culture plate by spinning a
        titanium-tipped pen into the plastic.

    b.  Cover each well with 50 μl of M16.

    c.  Overlay with mineral oil that is saturated with PBS.

    d.  Incubate plates at 37°C in 5% CO~2~ for at least 12 hr.

Day 6

1.  Prepare other plates that are required to generate aggregation
    chimeras and place them at 37°C in 5% CO~2~ for 1 hr.

    a.  Two 15 x 100 mm plates containing 15 ml of phosphate-buffered
        saline (PBS).

    b.  Two 10 x 35 mm plates containing 2 ml of M2.

    c.  Two 10 x 35 mm plates containing 2 ml of Tyrode's solution.

    d.  Ten 15 x 60 mm plates containing five 50 μl drops of M2.

2.  Isolate embryos from female donors.

    a.  Sacrifice female donors by CO~2~ asphyxiation. Remove uterus and
        ovaries. Place tissue in a dish containing PBS.

> ![](media/image22.png){width="3.263888888888889in"
> height="1.9444444444444444in"}

b.  The following manipulations are performed under a dissecting
    microscope.

c.  Cut off each ovary at the junction with the oviduct. Make a second
    cut to remove all but 5 mm of the uterus. Place oviduct in a dish
    containing 1 ml of M2.

d.  Blow embryos out of the oviduct. Attach a 30-gauge needle to a 1 ml
    syringe and fill it with 500 μl of pre-equilibrated M2. Hold the
    infundibulum with dissecting forceps and insert the 30-gauge needle.
    Flush out the embryos. You should see the oviduct expand and a
    stream of particles including embryos swirl out of the uterus.
    Locate embryos under high magnification. Transfer 4 to 16 cell
    embryos to a plate containing five drops of M2 using a 2-10 μl
    pipetman or a glass pipet.

```{=html}
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```
3.  Treat embryos with pre-equilibrated Tyrode's solution to remove the
    zona pellucida.

    a.  Transfer a few embryos from M2 into Tyrode's solution using a
        pipetman.

    b.  Move embryos around in order to dilute M2 medium in which they
        were transferred.

    c.  Remove embryos as soon as the zona pellucida disappears.

    d.  Transfer embryos to a plate containing 5 drops of M2.

    e.  Wash embryos by moving them through the drops of M2.

4.  Repeat all steps with female donors from the second strain.

5.  Generate aggregation chimeras.

    a.  ![](media/image23.png){width="2.9166666666666665in"
        height="0.8333333333333334in"}Transfer embryos using a glass
        pipet from M2 to M16 plates with wells such that there is one
        embryo from each strain per well.

    b.  Make sure that embryos are touching in the wells. Recheck after
        a couple of hours as embryos will often float apart or out of
        the wells.

Day 7

1.  Blow aggregation chimeras out of the wells early in the morning and
    transfer to M2 just before implantations.

2.  Implant aggregation embryos into the uterus of pseudopregnant ICR
    recipient. We usually implant 6 aggregation chimeras per side.

3.  ![](media/image24.png){width="2.625in"
    height="1.6388888888888888in"}Anesthetize pseudopregnant ICR
    recipient.

4.  Shave the lower back.

5.  Make an incision through the skin and then locate the ovary, which
    will be bright red.

6.  Make a second incision through the body wall.

7.  Pull the fat pad attached to the ovary and place the ovary on a
    small piece of Wypall. A small clamp can be attached to the fat pad
    to secure ovary and uterus.

8.  Grasp the uterus near the top with a pair of dissection forceps and
    puncture it with a 26-gauge needle. It is important to make sure the
    needle passes through the uterine wall.

9.  With the forceps as a landmark to find the hole, transfer embryos
    from M2 into the uterus with a glass pipet.

10. Slide the ovary and uterus back into the recipient and close the
    incision with wound clips. The body wall and skin can be closed
    simultaneously.

11. Wrap the recipient in a Wypall to conserve body heat.

Days 24-26

> Aggregation chimeras will be born. We usually foster the pups, because
> mothers often kill their pups.

## [Notes]{.underline}

1.  Superovulation is done in order to set the timing of the experiment
    rather than to increase the yield of embryos. Females must be
    treated with hormones between 21 and 28 days of age before they
    begin their own cycle. The amount of hormone that is effective is
    dependent upon the strain. We use 5 I.U. of PMS and 5 I.U. of HCG
    for B6 females.

2.  Injection of hormones needs to be intraperitoneal.

3.  BSA must be added to media to prevent embryos from sticking to the
    plates.

4.  Minimize the time that embryos and media spend out of the incubator.

5.  M2 is more stable than M16 at room temperature.

6.  Mineral oil is saturated with PBS by mixing an equal volume of both
    liquids. Mineral oil is less dense than PBS, so it will be the top
    layer. We have used just mineral oil.

7.  Extended treatment of embryos with Tyrode's solution will kill them,
    so treat only a few embryos at a time.

8.  Glass pipets need to be siliconized to prevent embryos from
    sticking. They are drawn out over an open flame to reduce the
    internal diameter. The opening of the glass pipet should be about
    twice the size of an embryo.

9.  Aggregation chimeras must be blown out of the wells before they
    progress to blastocysts, as it is impossible when they reach this
    stage of development.

10. If multiple ICR recipients are plugged by a single vasectomized
    male, then these mice should be used for implantations only as a
    last resort.

11. Some researchers keep pseudopregnant ICR recipients on a heating pad
    during implantations. We have found this practice not necessary.

12. Inject first bubble and embryos into the uterus. Limit the amount of
    air as it can interfere with implantations.

13. Aggregation chimeras are fostered to normal ICR females because
    mothers often kill their young. It is not clear why the
    pseudopregnant mothers kill the young.

####### Supplies

  ------------------------ ------------ ---------- ----------- -------------
  Item                     Amount       Cost       Source      Catalog No.

                                                               

  PMS                      1000 I.U.    \$44.20    Sigma       G4877

  HCG                      10000 I.U.   \$46.90    Sigma       CG-10

  M2                       50 ml        \$10.30    Sigma       M7167

  M16                      50 ml        \$10.30    Sigma       M7292

  BSA                      10 g         \$50.50    Sigma       A3311

  Mineral oil              500 ml       \$15.00    Sigma       M8410

  Tyrode's solution        100 ml       \$15.50    Sigma       T1788

  Penicillin G             1,000,000 U  \$9.40     Sigma       P4687

  Steptomycin sulfate      5 g          \$11.80    Sigma       S1277

  15 x 100 mm plates       Case of 100  \$93.60    Fisher      08-757-13

  15 x 60 mm plates        Case of 200  \$103.17   Fisher      08-772-4C

  10 x 35 mm plates        Case of 200  \$89.87    Fisher      08-772-4A

  Forceps, microdissecting 1            \$26.95    Roboz       RS-5130

  Scissors,                1            \$43.27    Roboz       RS-5910
  microdissecting                                              

  Clamp                    1            \$30.90    Roboz       RS-7423

  Pipet, unopette          Box of 100   \$61.10    Fisher      1368014
  ------------------------ ------------ ---------- ----------- -------------

[Examples]{.underline}

# 

![](media/image25.png)