Flow Cytometric Analysis Of Genotoxicity
Award last edited on: 6/17/08

Sponsored Program
Awarding Agency
Total Award Amount
Award Phase
Solicitation Topic Code

Principal Investigator
Johnathan C Sharpe

Company Information

Dakocytomation Colorado Inc (AKA: Cytomation GTX Inc)

4850 Innovation Drive
Fort Collins, CO 80525
   (970) 226-2200
Location: Single
Congr. District: 02
County: Larimer

Phase I

Contract Number: 1R43CA091566-01
Start Date: 00/00/00    Completed: 00/00/00
Phase I year
Phase I Amount
The objective of this proposal is to develop a low cost, rapid, reliable and sensitive flow method to determine mutagenesis. The system is based on a CHO AL cell line, which has human chromosome 11. Mutations in chromosome 11 are detected by loss of expression of surface antigens encoded on chromosome 11. The hypothesis is that mutations in chromosome 11 can be accurately, sensitively and rapidly detected by flow cytometry based on the loss of surface antigens. The specific aims of this proposal are to develop optimal methods for staining CHO AL cells with antibodies to measure the presence or absence of S1 antigen: determine the sensitivity of the flow cytometry mutagenesis assay; validate the assay by comparing mutant fractions measured with flow cytometry to that found by the standard assay; and identify design parameters for a small, reliable inexpensive, high resolution, low noise, fast flow cytometer to make routine measurements of mutant frequency. The development of this methodology would allow for rapid and inexpensive screening pharmaceuticals for genotoxicity

Phase II

Contract Number: 2R44CA091566-02
Start Date: 00/00/00    Completed: 00/00/00
Phase II year
(last award dollars: 2003)
Phase II Amount

The overall objective of this proposal is to develop a flow cytometry- based assay utilizing optimized flow cytometry instrumentation and cultured mammalian cells that can be routinely employed by pharmaceutical and other companies to measure the mutagenic potential of chemicals and drugs. The assay is based on a Chinese hamster ovary- human hybrid cell line, CHO AL, which contains human chromosome 11. The principal target for mutation is the gene for the CD59 antigen which is encoded on chromosome 11. The hypotheses are(1) that mutations in chromosome 11 can be detected accurately, sensitively and rapidly by flow cytometry measurement of the loss or presence of surface antigens encoded by chromosome 11 and (2) that this mutation assay system can be developed so that it could be used routinely to determine the genotoxicity of physical and chemical agents. Specific Aim 1 is to build a relatively inexpensive benchtop flow cytometer optimized for analysis of antigen loss in mammalian cells. Specific Aim 2 is to validate the flow cytometry assay for detecting mutations in CHO AL cells by measuring the mutation dose response from a panel of 25 physical and chemical agents at a range of doses. Independent studies will be done at an off-site laboratory. Specific Aim 3 is to extend the assay to include the analysis of intragenic and chromosomal mutations by measuring the presence or absence of multiple antibodies bound to different cell surface antigens encoded by separate genes on human chromosome 11. The development of this assay system would allow for rapid, accurate, relatively cheap but informative screening of pharmaceuticals for genotoxicity and substantially reduce the cost of carrying out mutation analysis.

Thesaurus Terms:
DNA damage, flow cytometry, gene mutation, method development, surface antigen cytotoxicity, drug adverse effect biotechnology, cell line