Optics: measuring and testing – Inspection of flaws or impurities
Reexamination Certificate
1999-08-20
2002-06-04
Rosenberger, Richard A. (Department: 2877)
Optics: measuring and testing
Inspection of flaws or impurities
C209S639000
Reexamination Certificate
active
06400453
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present application concerns high-speed mechanisms for automatically identifying and physically selecting multicellular organisms or other large objects with predetermined characteristics from mixed populations and depositing them in discrete locations.
2. Description of Related Art
Intact multicellular organisms, such as nematodes, fruit fly larvae, or zebrafish embryos are frequently used as model systems to help understand the function of human genes that have been implicated to play a role in disease. Human gene homologous have been identified in these model organisms and mutations have been induced specifically in those gene homologous. Such mutations frequently result in an easily observable phenotypic change in the model organism, and it has been shown that certain mutants respond to pharmacological compounds with a measurable mode of action. Mutants of intact organisms are now used as a new class of in vivo drug screens for combinatorial pharmacological compound libraries. By using these organisms, one can identify targets for drug intervention without the need to completely understand complex biochemical pathways between the genotype and the phenotype. In addition solid state combinatorial chemical approaches are now being utilized to produce these drug libraries; the end result is that the sample chemicals to be tested are present on solid microspheres usually between 100 and 500 &mgr;m in diameter. These solid state techniques greatly speed the preparation of the sample compound library but necessitate a method to accurately select and dispense these microspheres for testing purposes.
The historic approach to modeling diseases in multicellular organisms has been to make morphological or behavioral mutants with substantial phenotypic defects. The intent of such research is to produce a mutant that resembles or models a disease state so that new therapeutics can be screened without using human “guinea pigs.” In fact, considering the current prevalence of animal rights activists, the safest approach is to entirely eschew the use of mammals for testing purposes. The goal, then, has been to observe these model disease defects and their interaction with candidate therapeutics objectively and with high sensitivity. Unfortunately, this goal has been not often met. The closest approach to reaching the goal has been to devise “live-dead” assays that can be carried out in microwell arrays using optical readout systems. The plan is to dispense individual organisms into microwells, add the candidate therapeutic and optically detect the response. If the candidate therapeutic is present on a microsphere, then the microsphere must also be accurately selected and dispensed.
The exposure of model organism mutants to diverse pharmaceutical compound libraries, even when the mutation has not been linked to a human gene homologue also helps define gene function. The addition of such functional genomic techniques to the repertoire of molecular biology and biochemistry methods is leading to a significant increase in speed in the pharmaceutical discovery process. Investigators annotate pharmaceutical drug libraries for toxicity, non-specific activity, or cell membrane permeability, etc. by observing their behavior in intact organisms. This way, potential new therapeutics that show toxicity or harmful results can be discarded early without wasting valuable resources.
The soil nematode
Caenorhabditis elegans
, has become a particularly important multicellular organism for these types of tests because its anatomy, development, behavior and genome, is more completely understood than that of any other animal.
C. elegans
is a small metazoan animal composed of only 959 cells, each generated from a single zygote cell through a completely known cell lineage. This small number of cells nonetheless exhibits a diversity of cell types that typifies more complex animals, including skin, muscle, gut and nerve cells.
The genes of
C. elegans
are easily accessed through powerful classical and molecular genetic tools. The sequencing of the
C. elegans
genome is also more advanced than that of any other animal and is a model for the Human Genome Project. Although most human disease genes that have been identified and cloned based on chromosomal position have no known function, the vast majority of these as well as most other human genes have
C. elegans
homologs. These homologs can be rapidly analyzed using the above-mentioned approach to elucidate the functional biology of the homologous human gene.
A striking conclusion from studies of
C. elegans
is that the cellular and molecular mechanisms that operate in this nematode are strikingly similar to those that operate in more complex animals, including man. These similarities are so great that homologous human genes can function in nematodes and nematode genes can function in mammalian cells. Researchers are therefore using this nematode for numerous types of experiments related to the development of pharmaceutical agents for use in humans and other higher animals.
Despite the potential power and speed of using multicellular organisms like
C. elegans
current programs for rapid pharmaceutical drug discovery of not employ high-speed preparation techniques. As an example, with today's molecular biology techniques, a large laboratory can produce deletion mutations in multicellular organisms at a rate of 20 to 30 per month. To evaluate the effect of a chemical compound library (that frequently may contain 100,000 or more members) on a class of mutated organisms, one must first manipulate and deposit a precise number of organisms in the same development stage into a container, such as the wells of a microtiter plate array. Organisms of different development stage must be excluded since they would convolute the measured response.
Using slow, manual methods, the selection and deposition of organisms of the proper type is a bottleneck for the entire process of pharmaceutical discovery. If the test compounds are present as microspheres, then the accurate selection and dispensing of microspheres adds an additional bottleneck. Furthermore, manual methods rely on pipettes that dispense accurate volumes of fluid and not accurate numbers of organisms. In many studies where reproduction rate is altered by the mutation, it is necessary to begin the study of the effect of a compound from the combinatorial library with an exact, and known number of multicellular organisms in each well. Any selection system based on volume is liable to dispense inaccurate numbers of organisms because precisely uniform suspensions of organisms are impossible to maintain. In the same way if the test compounds are available as microspheres it is extremely difficult to place a controlled number of microspheres in each well. Further, the microsphere population may be mixed so ultimate results require not only precise counting but selection of microspheres—clearly an impossible task for simple pipettes.
Flow cytometers have operational characteristics that make them adaptable to the problems of automating the selection and deposition of multicellular organisms and other large objects such as microspheres. Flow cytometers have been used to count the number of nematodes in a given volume of fluid. Such a device was described by Byerly et al (Byerly, L., R. C. Cassada, and R. L. Russell, “Machine for Rapidly Counting and Measuring the Size of Small Nematodes”, Rev. Sci. Instrum. Vol 46, No. 5, May 1975) where the flow cytometer utilized sheath flow to orient the nematodes along the direction of flow so that their length could be measured and organism-by-organism counts could be made by an electrical impedance method similar to that used in a commercial Coulter® counter. A flow cytometer for working with multicellular organisms is not limited to using an impedance sensor, biut can be a more modern optically sensing flow cytometer.
For example, an optical flow cytometer for analyzing elongate organisms such as plankton with widths of
Crosby Heafey Roach & May LLP
Kirchanski, Esq. Stefan J.
Rosenberger Richard A.
Union Biometrica, Inc.
LandOfFree
Instrument for selecting and depositing multicellular... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Instrument for selecting and depositing multicellular..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Instrument for selecting and depositing multicellular... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2922737