Primate samples can be submitted to the Genetics Core in order to establish viable cell lines and for storage for the eventual isolation of DNA. The following procedures have been developed to help investigators and staff to properly submit samples to the Genetics core. The Genetics core will also store frozen tissue samples per investigator request.

• Fibroblast Biopsy: SOP 001
• Tissue Collection: SOP 002
• Blood sample: SOP 003
• Submission of samples for Karyotype Analyses: SOP 004
• Procedure to Ship samples to the CNPRC Genetics Core: SOP 005

Fibroblast Biopsy
SOP 001

This SOP pertains to all monkeys passing through necropsy and animals undergoing surgical procedures, with investigator approval. Viable cell lines from fibroblasts will be established to provide a nearly inexhaustible source of DNA and to provide a viable cell line for in vitro research.

Supplies Requires:

Tissue culture media or sterile saline (Room temperature) for biopsy transport
Clippers to remove fur
70% alcohol
sterile gauze
4 mm biopsy punches
forceps
suture or skin adhesive
suture forceps
marker
sedation supplies

Contact the Genetics Core to alert investigators of biopsy collection: 754-7087. This phone will forward to Dr. Lyons' lab in Tupper Hall in no one is available in Room 1218.

1) Obtain tubes (15 ml conical) of sterile tissue culture media from the Genetics Core (Room 1218) or from the refrigerator in necropsy. Media can be stored for 4 weeks at 4 C, but should be at room temperature when the biopsy is placed in the media. More than one biopsy can be placed in a tube, but it must be from the same animal. The media contains antibiotics and nutrients. Sterile saline can be used if media can not be obtained.

2) Label tubes with the date, sex, and monkey ID.

3) Properly sedate live animals. Non-living animals can be biopsied if death has occurred within 1 - 2 hours.

4) Select a biopsy site that will cause minimal trauma to the animal and be the least likely to become infected. Currently, the area between the scapulas is suggested. For non-living animals, any area can be selected for biopsy. In an animal is undergoing a surgical procedure, the biopsy can be taken as a thin strip of skin from the incision site.

5) Remove the fur from the biopsy site. Excess fur should be removed to ensure an aseptic collection. Clip as close to the skin as possible.

6) Clean the area with water and 70% alcohol. Please DO NOT use betadine or Nolvasan. These chemicals will interfere with proper cell growth. The area should be scrubbed if required. Fungus from the skin and fur is a very frequent contaminant and will destroy the tissue culture.

7) Collect the biopsy with a 4mm skin punch or with forceps and scissors. The biopsy needs to be the full thickness of the dermal layer. The biopsy does not need to be larger than 4 mm, but larger samples can be a benefit. Skin from larger samples can be viably frozen for future culturing. Thus, non-living animals would be strong candidates for larger biopsies.

8) Place biopsy in the media/saline immediately. Keep the samples at room temperature or lower.

9) If the biopsy gets contaminated or dirty (dropped on the floor or ground), rinse it with 70% alcohol and place in the media.

10) Samples need to be taken to the Genetics Core and placed on the counter in Room 1218 for culture set-up. Culture set-up needs to occur within 12 - 24 hours.

11) Please report any occurrences of biopsy site infection or other complications to the core.

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Tissue Collection
SOP 002

This SOP pertains to all monkeys passing through necropsy, with investigator approval. Tissues will be collected from dead or sacrificed animals for DNA isolation. Tissues will include spleen, kidney and mesenteric lymphnodes.

Supplies Requires:

1.5 ml freezer tubes
liquid nitrogen

Contact the Genetics Core to alert investigators of tissue collection: 754-7087. This phone will forward to Dr. Lyons' lab in 1121 Tupper Hall in no one is available in CRPRC Room 1218.

1) Labeled tubes (1.5 ml cryovials) needed to be obtained from the Genetics Core. Tissue tubes should be labeled with the date, ID, tissue type, and sex if not available from the core.

2) See also Genetics Core SOP 001 "Fibroblast Biopsy" for recently deceased or euthanized animals.

3) During necropsy, tissues should be collected and snap frozen in liquid nitrogen as soon as possible.

4) Samples should be collected from spleen, kidney, and peripheral lymphnodes. Mesenteric lymphnodes are suggested.

5) The tissue needs to be sectioned into portions that will fit into the 1.5 ml cryovials. Tissue can also be saved in baggies or foil if tubes are not available.

6) Six samples will be collected from each tissue. If the cryovials were not available, section the tissue into six small pieces (about the size of an enlarged lymphnode).

7) Place the tissues in the appropriately labeled tube and snap freeze with liquid nitrogen.

8) Once sampling is complete, contact the Genetics Core for pick-up or samples can be placed in the core freezer in Room 1218.

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Blood Sample Collection
SOP 003

This SOP pertains to all monkeys that are sedated for routine or non-routine health evaluations, any animals passing through necropsy and animals undergoing surgical procedures, with investigator approval.

Anti-coagulated blood samples will be collected to establish a WBC-based DNA resource for all primates at the CRPRC.

Plasma, RBCs, WBCs, and DNA from WBCs will become available resources.

Supplies required:

10ml EDTA (purple top) blood tubes
ice
appropriate needles, syringes,
alcohol

Contact the Genetics Core to alert investigators of blood sample collection: 754-7087. This phone will forward to Dr. Lyons' lab in 1121 Tupper Hall in no one is available in CRPRC Room 1218.

1) Obtain labeled tubes from the Genetics Core: CRPRC Room 1218. Blood tubes should be labeled with the date, ID, and sex if not available from the core.

2) Select an appropriate collection site for venous blood collection.

3) Collect approximately 10 mls of venous blood. The animals size and health status should be considered for the amount of blood collected.

4) Place the blood samples on ice or keep the sample at room temperature. Do not freeze the sample.

5) Deliver samples to the Genetics Core or call the core for retrieval of the samples. Samples can be placed in the core refrigerator in Room 1218.

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Karyotyping Service
SOP 004

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Shipment of Samples to the CRPRC Genetics Core
SOP 005

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