Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2002-05-15
2004-10-12
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S004000, C435S040500
Reexamination Certificate
active
06803207
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an apparatus and a method for selecting oocytes or eggs. It also relates to an oocytes or egg population having a specific level of membrane potential and to a method of selling or transferring the same.
2. Description of Related Art
Oocytes have been widely used for the purpose of introducing thereinto a gene, dye, protein, peptide, drug or like sample to confirm the action, analyze the genetic function, or producing the gene product protein, for instance.
The main criterion employed in the art for selecting oocytes or eggs is the appearance. The criteria for selecting Xenopus laevis (African clawed toad) oocytes are, among others, that the diameter is 1.2 mm, that the shape is perfectly spherical, that the boundary between the black surface and white surface is distinct, and that there is no mottle or spot. The selection work is carried out manually under a microscope.
The selection of oocytes or eggs based on the appearance has the problem that the oocytes or eggs sorted out may fluctuate in quality depending on the ability and skill of each individual technician. One of the causes is that the selection criteria are rather ambiguous and discretionary. In the prior art, the oocytes or eggs sorted out can hardly be ranked. Thus, for example, the survival rate, the fertilization rate, the efficiency in introducing a sample, such as a gene, vary from cell to cell. Further, it is difficult to correct or control the fluctuations among parents in terms of survival rates, fertilization rates and gene transfer efficiency, for instance. As a result, it is only by chance that cells show no great fluctuations in survival rates, fertilization rates, and gene transfer efficiency. It is an object of the present invention to formulate certain oocyte or egg selection criteria numerically.
Furthermore, in the prior art, no due consideration has been given to the storage of data on the cells selected. Thus, it is difficult to establish correlations between data regarding selection standards and later cellular responses. Accordingly, it is another object of the invention to find such correlations.
Since, in the prior art, oocytes have been selected manually, mass production is impossible.
Accordingly, it is a further object of the present invention to produce, sell, and transport oocytes or eggs guaranteed being selected according to an established standard.
SUMMARY OF THE INVENTION
In order to accomplish the above objects, the present invention provides an apparatus and method for selecting oocytes or eggs which comprise, or comprise the use of, measuring means for selecting oocytes or eggs and selecting means for sorting the oocytes or eggs based on the measurement results.
Thus, the present invention provides a method or mechanism for selecting oocytes or eggs having a certain size out of a plurality of oocytes or eggs by means of a filter or the like as well as a step or mechanism for measuring the plurality of oocytes or eggs for membrane potential and sorting out those having a certain membrane potential from among them. In this manner, it provides oocytes or eggs uniform in quality.
The present invention first provides an apparatus for selecting oocytes or eggs which comprises measuring means for measuring the membrane potential of oocytes or eggs, and selecting means for selecting the oocytes or eggs based on the membrane potential measurement results. By this, it obtains oocytes or eggs with a certain specific level of membrane potential.
The present inventors discovered certain correlations between the membrane potential and the appearance characteristics, ex. the shape, the distinctness of the boundary between the black surface and white surface, and the presence or absence of mottles or spots, of various oocytes or eggs, especially Xenopus laevis oocytes. More specifically, they found that those oocytes, which are nearly spherical, have a distinct boundary between the black and white surfaces and are free of any mottle or spot, and have a membrane potential maintained at about −70 mV shortly after isolation. On the other hand, those cells, which are rather non-spherical, show an unclear boundary and/or have a mottle(s) or spot(s), also have a shallower membrane potential (larger negative membrane potential, hence smaller absolute potential value). By utilizing the findings and measuring the membrane potential, the invention does without the prior art appearance observation step in a selection process.
The preferred methods for measuring the membrane potential include a method based on the technique of intracellular recording or a voltage sensitive dye method. The voltage sensitive dye method comprises injecting a dye into (staining) a cell in advance, and detecting the change in the wavelength of light absorbed by the dye according to the change in the membrane potential.
The intracellular recording method comprises measuring the potential difference between the inside and outside of the cell membrane. In carrying out this method, one electrode is disposed outside the cell membrane and a second electrode inside the cell membrane, then the potential difference between them is measured. The second electrode is inserted into the cell to a depth of about 0.1 &mgr;m −0.5 mm form the cell surface.
Furthermore, the inner of the container to be used in the membrane potential measurement has a metal coating for grounding such that the measurement can be performed simply and rapidly.
The present inventors further discovered that when a gene is introduced into oocytes, the protein function expression efficiency varies depending on the membrane potential of the oocytes (before gene introduction). Thus, for preventing the protein function expression efficiency from fluctuating among oocytes, it is important to select oocytes having the same membrane potential. The present invention provides an apparatus for selecting oocytes or eggs comprising means for selectively collecting oocytes or eggs having a specific level of membrane potential. By this, it has become possible to select oocytes or eggs having a specific level of membrane potential.
Furthermore, the present invention provides an apparatus for selecting oocytes or eggs comprising measuring means for measuring the sizes of oocytes or eggs and selecting means for selecting the oocytes or eggs based on the size measurement results. By this, it has become possible to select those oocytes or eggs with a specific size. In this case, it is preferable to provide a storage medium for storing the membrane potential data of the oocytes or eggs such that the selecting means can select based on the stored information.
For collecting oocytes or eggs with a specific size, it is preferable to use a filter having a definite pore size. Since oocytes have a diameter of about 1.2 mm, the filter pore size is preferably not more than 1.4 mm. The filter may have honeycomb-like pores or round pores. A filter having a plurality of pores with a diameter of 1.0-1.4 mm may be used to sort out oocytes or eggs having a diameter within a certain desired range.
The present invention further provides an apparatus for selecting oocytes or eggs comprising means for selecting oocytes or eggs with a specific size and means for measuring the membrane potentials of the oocytes or eggs. By this, it becomes possible to select oocytes or eggs whose size and membrane potential are within respective specific ranges.
The invention further provides a tray for measuring the membrane potentials of oocytes or eggs which is characterized in that it has a container for receiving an oocyte or egg and that the inside of the container has a metal coating for grounding. The method for selecting oocytes or eggs further comprises a measuring step or steps for selecting oocytes or eggs.
The method according to the present invention comprises the step of measuring the membrane potentials of oocytes or eggs or the step of measuring the sizes of oocytes or eggs, or both the steps. By this method, th
Matsunami Shokichi
Otomo Jun
Takeshita Tomoko
Davis Ruth A.
Lankford , Jr. Leon B.
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