Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-09-14
2002-05-07
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S285100, C435S455000
Reexamination Certificate
active
06383813
ABSTRACT:
BACKGROUND OF THE INVENTION
Transgenesis, the introduction of foreign gene(s) into a mammalian genome, has its beginnings in the recombinant DNA technology discovered by Boyer and Cohen. By creating the basic tools necessary to directly manipulate DNA, specific genetic modifications at the organismal level was possible. These tools were first applied to simple, unicellular bacteria to both introduce foreign genes and remove endogenous genes but it was quickly realized that these tools were equally applicable to potentially more useful multicellular organisms—e.g. mice, pigs, sheep, goats, and cows. The ability to create transgenic organisms by inserting or deleting specific genes allows one to specify the phenotypic characteristics of the organism—a powerful tool with potentially limitless applications, both commercial and scientific.
Cloning already has demonstrated commercial value, particularly in the production of living “bioreactors”, i.e.—transgenic animals expressing therapeutic, pharmaceutically valuable human proteins secreted in milk or urine. As cloning becomes routine, more speculative applications such as transgenic pigs appropriate as a source of organs for xenotransplantation become feasible and potentially could alleviate the current shortage of human donor organs. Moreover, in addition to biomedical applications, transgenic technology will likely have agricultural applications that directly benefit the everyday consumer by improving the disease resistance, growth rate, feed efficiency, and nutritional quality of commercial livestock
The recent announcement of Dolly, a sheep cloned from an adult somatic cell, and confirmation of the result in mice, cows, and pigs, has generated intense excitement in the potential of cloning as a greatly improved method for generating transgenic animals. Cloning requires nuclear transfer—the nucleus of the recipient oocyte is removed (“enucleation”) and replaced by the nucleus of a donor cell, i.e. the donor nuclei is transferred into a recipient oocyte and the “reconstructed” embryo that develops has the characteristics of the donor individual While nuclear transfer has numerous advantages over pronuclear injection (currently, the most popular method), it is limited by the extremely low efficiency of generating viable offspring (~1-2%)—in a typical series of nuclear transfers, one hundred oocytes result in at most one or two live births which develop into adults. This low efficiency places severe limitations on the commercial use of cloning, especially in domestic animals which have long gestation periods. In addition, nuclear transfer is very technically demanding—microinjection of eggs requires precise manipulations under a high power microscope using expensive micromanipulators and success is often quite dependent on the skill of the operator. Clearly, if the efficiency and/or throughput of nuclear transfer could be improved, the commercial applications become much more feasible and cost-effective.
The low efficiency appears to occur at two points in the nuclear transfer procedure. Following nuclear transfer, only ten to twenty percent of the reconstructed embryos survive to a cell stage that allows them to be implanted in a host. Once implanted, fetal development is often abnormal and most embryos (80-90%) either abort or are stillborn. Improving the low survival rates will require significant research; however an immediate solution is obvious. Currently, nuclear transfer is done serially—i.e. eggs are manipulated individually. If, for example, a thousand or more nuclear transfers could be done simultaneously, the throughput of nuclear transfer increases, minimally, one thousand-fold. Eighty to ninety percent of these embryos, as before, would not survive, but in a given time period the absolute number of implantable embryos (and consequently, transgenic animals) dramatically increases.
SUMMARY OF THE INVENTION
To achieve improved throughput a unique approach which integrates cellular nuclear transfer within a microfabricated silicon-based bioarray has been devised. This nuclear transfer array (NTA) consists of hundreds or thousands of individual nuclear transfer units which, in parallel, will perform the enucleation, transfer, and insertion steps necessary to accomplish nuclear transfer. The advantages of this approach to nuclear transfer will significantly advance adoption of nuclear transfer as a standard technology for producing commercially important transgenic animals and also Father progress in realizing the vast potential of animal cloning in biomedical and agricultural applications.
A micro-machined array (nuclear transfer array, NTA) which allows high-throughput transfer of nuclei between two cells is designed to increase the success of nuclear transfer. A silicon or glass substrate is patterned with parallel rows of cylindrical microwells of the diameter of the cell of interest. A hole is etched in the bottom of the microwell to form an “injection port”. This array of microwells forms the top “enucleation” component of the complete NTA. To accomplish nuclear transfer, a bottom or “re-nucleation” component is also necessary. This consists of a second array of microwells of similar dimensions and in register with the top enucleation component. In addition, the re-nucleation component is manufactured with a gasket and outlet which allows vacuum suction to be applied to the complete array. An individual nuclear transfer unit is comprised of the upper enucleation microwell (“upper chamber”), the injection port, and the lower re-nucleation microwell (“lower chamber”). Hundreds to thousands of these nuclear transfer units can be patterned into an individual array.
Individual eggs, oocytes or biological cells are placed into the microwells of the upper chamber. The cells are drawn towards the injection port by centrifugation on a Percoll gradient which centers the nucleus of the cell directly over the injection port and adjacent to the cell membrane. Suction is preferably applied, the membrane is pierced and the nucleus is removed. Nuclei are collected in a lower chamber. Alternatively, nuclei can be purified from cells grown in culture and placed in the wells of the lower chamber. The upper chamber—containing alternating rows of either recipient biological cells or donor biological cells—moves relative to the lower chamber, containing the extracted nuclei. In any case, following enucleation and movement of the upper chamber relative to the lower chamber, the recipient biological cells are positioned in the upper chamber above a donor nucleus. The suction is then reversed resulting in recipient biological cells containing donor nuclei. Alternate means may be used to transfer the nuclei between the upper chamber and lower chamber, including applying electric fields, magnetic fields and centrifugal forces.
The NTA offers a number of advantages over prior nuclear transfer techniques. The NTA is a highly parallel operation such that hundreds to thousands of nuclear transfer (cloning) operations can be done simultaneously with significantly greater throughput than previously possible. The process is automated and therefore should be more reproducible. Most important, the NTA provides a solution to the low efficiency of the cloning process because the absolute number of clones is no longer limiting.
The NTA should be broadly applicable to any use envisioned for cloning. Some applications include producing transgenic animals which express therapeutic, pharmacetically useful proteins in their milk, generation of non-immunoreactive transgenic pigs for xenotransplantation, development/expansion of superior quality livestock (i.e. higher quality meat, wool, milk, etc.), and individualized creation of human stem cells for replacement therapy. In addition, cloning addresses important scientific questions regarding genomic differention to cancer and aging, and potentially allows the creation of genetic organisms in a more efficient manner and in species other than the mouse.
REFERENCES:
patent: 4247646 (1981-01-01), Berky et al.
pa
Baxter Gregory T.
Kuo Richard C.
Cornell Research Foundation Inc.
Jones Tullar & Cooper PC
Redding David A.
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