U.S. Army Medical Research and Materiel Command (MRMC)

DESCRIPTION: Mice are an inexpensive, convenient animal model to test the efficacy of OP protectants and antidotes. However, unlike humans and non-human primates, mice express a serum carboxylesterase enzyme (Es-1) (1-4); due to the capacity of Es-1 to act as an endogenous bioscavenger of OPs, mice are substantially more resistant to OP toxicity than primates (5). We propose to generate genetically modified mice in which the gene encoding the Es-1 serum carboxylesterase has been inactivated by homologous recombination (6-8). Es-1 knockout mice are expected to have similar susceptibility to OPs as primates, but at a dramatically reduced cost per animal. Further, results from these Es-1 deficient mice will be more directly relevant to humans than those from existing small animal models.

PHASE I: The generation and characterization of Es-1 knockout mice requires the assembly of a homologous recombination construct encoding a version of the Es-1 gene (6-8) interrupted by stop codons (making the Es-1 gene non-functional). This construct will then be transfected in an embryonic stem cell line, and recombinant stem cells will be implanted into a mouse uterus (in mixed normal/recombinant blastocysts) where they will develop into chimeric offspring mice. Chimeric animals will be screened for germ line transmission of the interrupted, non-functional Es-1 gene in order to generate heterozygous knockout mice. Finally, heterozygous Es-1 knockouts will be bred to produce homozygous knockout mice that lack a functional copy of the Es-1 gene, and thus will be unable to produce serum carboxylesterase. These mice will then be examined for carboxylesterase activity in the serum, and for increased susceptibility to OPs (proof of concept). Although unlikely, it is possible that elimination of Es-1 from the serum will have adverse effects on mice, including reduced viability or infertility. If such problems arise, heterozygous Es-1 knockout mice (which retain one functional copy of the Es-1 gene) will be analyzed for OP susceptibility. We are capable of supplying the necessary DNA constructs for development of chimeric animals, but an industrial partner capable of the necessary manipulation of stem cells, and subsequent development of chimeric animals is required to accomplish the goal of this project. These techniques are in most cases not yet standardized or routine, and we will defer to the experience and advice of the small business partner with respect to experimental protocols and approaches.
PHASE II: After large-scale mouse breeding to produce a sufficient working stock, OP-neutralizing bioscavengers such as mutated butyrylcholinesterase (developed in-house at USAMRICD) will be tested for their ability to protect Es-1 knockout mice against OP toxicity. Additionally, the efficacy of other anti-OP interventions such as anti-convulsants and acetylcholinesterase reactivating agents will be tested using the Es-1 knockout mice model system.

PHASE III DUAL USE APPLICATIONS: After fully characterizing Es-1 knockout mice and establishing their utility as a animal model system for studying pretreatments and antidotes for OP exposure, these mice will be made available to researchers and companies working in related areas. For commercial applications, these mice will present a financially viable animal model for the study of pesticide toxicity and the efficacy of treatments for pesticide exposure. In primary research fields, Es-1 knockout mice will be of interest to researchers studying carboxylesterase pharmacology and metabolism, as well as the metabolism of xenobiotic compounds.

OPERATING AND SUPPORT COST (OSCR) REDUCTION: Current cost estimates for the purchase and per diem housing of a rhesus monkey are $4000 and $4.79 per day, respectively. Es-1 knockout mice will be bred in our animal facility (no purchase cost), and the per diem for a mouse is only $0.74 per day. Thus, the use of Es-1 knockout mice in place of rhesus monkeys in appropriate experiments will generate a substantial operating cost reduction while still fulfilling research mission objectives.

REFERENCES:

1. Ovnic, M., Tepperman, K., Medda, S., Elliott, R.W., Stephenson, D.A., Grant, S.G. and Ganschow, R.E. (1991) Characterization of a Murine cDNA Encoding a Member of the Carboxylesterase Multigene Family. Genomics, 9, 344-354.

2. Medda, S. and Proia, R.L. (1992) The Carboxylesterase Family Exhibits C-terminal Sequence Diversity Reflecting the Presence or Absence of Endoplasmic-reticulum-retention Sequences. Eur. J. Biochem., 206, 801-806.

3. Robbi, M. and Beaufay, H. (1992) Topogenesis of Carboxylesterases: A Rat Liver Isozyme Ending in -HTEHT-COOH is a Secreted Protein. Biochem. and Biophys. Research Comm., 183, 836-841.

4. Kadner, S.S., Katz, J. and Finlay, T.H. (1992) Esterase-1: Developmental Expression in the Mouse and Distribution or Related Proteins in Other Species. Arch. Of Biochem. And Biophys., 296, 435-441.

5. Maxwell, D.M., Brecht, K.M. and O'Neill, B.L. (1987) The Effect of Carboxylesterase Inhibition on Interspecies Differences in Soman Toxicity. Toxicology Letters, 39, 35-42.

6. Capecchi, M.R. (1989) Altering the Genome by Homologous Recombination. Science, 244, 1288-1292.

7. Galli-Taliadoros, L.A., Sedgwick, J.D., Wood, S.A. and Korner, H. (1995) Gene Knock-Out Technology: A Methodological Overview for the Interested Novice. J. Immunol. Meth., 181, 1-15.

8. Shastry, B.S. (1998) Gene Disruption in Mice: Models of Development and Disease. Mol. And Cell. Biochem., 181, 163-179.

A00-161 TITLE: Dry System for Thawing Frozen Blood

PHASE I: Design and fabricate a laboratory prototype warming device for thawing blood and preheating IV solutions. Identify a new blood bag material and an inexpensive shipping container. Do preliminary investigations on improving the deglycerolization protocol to reduce time and the amount of wash fluids.