Use of ALZET® Osmotic Pumps in Immunodeficient Mouse Models LJ\ “The combination of the established SCID mouse model for RA and the long-term osmotic pump-based delivery system for bioactive molecules adds a novel tool for the detailed analysis of RA pathophysiology.” (Knedla, A, et al. Ann Rheum Dis 2009;68:124-129) “The comparative experiments (injection and infusion) confirmed that continuous treatments and long-term administration were associated with the best treatment responses.” (Tejeda, M, et al. Anticancer Research 2006; 26(4B): 3011-3015) Immunodeficient mouse models have been an integral part of pre-clinical research, and have contributed to significant advances in oncology, transplantation, immunology and stem cell biology research. There are two main species of immunodeficient mice used in research today: SCID and Nude mice. SCID (severe combined immunodeficiency) mice lack the ability to make T or B lymphocytes due to a genetic mutation in chromosome 16. Nude mice also ^ undergo a genetic mutation (disruption of the FOXN1 gene) that leads to a lack of, or deteriorated thymus gland and inability to produce T lymphocytes. In addition to inhibiting their immune system, this mutation also causes a lack of hair growth in the animals, hence their name. Since these species do not exhibit normal immunological responses (e.g., fighting infections or rejection of tumors or transplants), they have been extensively used as biological hosts for tissue transplants since. It is this particular feature that makes these species extremely valuable in transplantation, oncology, and immunology research. ALZET Osmotic Pumps have been used extensively in immunodeficient mice since 1980, and over 250 publications are available as evidence of their research value in these species. The automatic operation and small size of ALZET pumps makes them an ideal infusion system for chronic dosing studies in Nude and SCID mouse models. No researcher intervention is required during infusion, and animal handling is kept to a minimum to reduce the risk of infection and stress. Nevertheless, since SCID and Nude mice are more delicate than normal mice, there are inherent concerns of using them in research studies involving experimental procedures, such as ALZET pump implantations. This technical sheet is intended to serve as a guide for successful use of ALZET pumps in SCID and Nude mice. dzef OSMOTIC PUMPS ALZET Osmotic Pumps I 10260 Bubb Road I Cupertino, CA 95014 T: 1 (408)-367-4036 F: 1 (408)-865-1406 Toll Free: 1 (800)-692-2990 www.alzet.com I [email protected]

COMMONCONCERNS, AND SOLUTIONS, WHEN USING ALZETOSMOTICPUMPS WITH IMMUNODEFICIENT MICE: • Pump size is too large. The smallest ALZETpump models (100X series) are only 1.5 cm in length x 0.6 cm in diameter. These pumps can be implanted subcutaneously in animals as small as 10 grams. They have widely been used in very small rodents (over 600 references), including neonatal rats and small mice. • Procedure is too invasive. The subcutaneous ALZET pump implantation is minimally invasive, and it is relatively quick and easy to perform. In fact, this procedure can typically be completed within a few...

MINIMUM ANIMAL WEIGHTS Implantation Pump Models Pump Models 1002, and 1004 2002, 2004, and 2006 Subcutaneous 10+ gram mice 20+ gram mice Intraperitoneal 20+ gram mice Via catheter Instrumentation • Autoclavable tip guards for surgical instruments should be used. • Special instrument trays with rows of soft plastic fingers can prevent damage of delicate instruments. • Check the tips of delicate instruments--preferably under a microscope--to make sure they are not damaged or dull, and check grooves to verify that no blood or tissue remains. • Do not use damaged or dull instruments. • Steam or dry...

Additional Tips/Methods Extracted from Recent Publications Mice were “allowed to acclimate to local conditions for at least 1 week” P8817 Dings, R.P.M, et al. Cancer Letters Technical 2008;265:270-280 Bulletin 1.1 “Alzet pumps were aseptically filled with the specific solutions, and implanted subcutaneously into the left lateral flank (opposite to the tumor) of the mice under anesthesia (ketamine 100 mg/kg and xylazine 3 mg/kg), and the incision was closed by wound clips.” Dings, R.P.M, et al. Cancer Letters 2008;265:270-280 “Animals were anesthetized with 2.5% isoflurane during imaging (BLI).” Zhang,...