News Column

Patent Application Titled "Cytoplasma to Organelle Delivery System and Associated Methods" Published Online

May 27, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventors Aten, Quentin T. (Orem, UT); David, Regis A. (Provo, UT); Burnett, Sandra H. (Saratoga Springs, UT); Jensen, Brian D. (Orem, UT); Howell, Larry L. (Orem, UT), filed on October 18, 2013, was made available online on May 15, 2014 (see also Brigham Young University).

The assignee for this patent application is Brigham Young University.

Reporters obtained the following quote from the background information supplied by the inventors: "Microinjection of foreign materials into a biological structure such as a living cell can be problematic. Various transfection techniques include the microinjection of foreign genetic material such as DNA into the nucleus of a cell to facilitate the expression of foreign DNA. For example, when a fertilized oocyte (egg) is transfected, cells arising from that oocyte will carry the foreign genetic material. Thus in one application, organisms can be produced that exhibit additional, enhanced, or repressed genetic traits. In some cases, researchers have used microinjections to create strains of mice that carry a foreign genetic construct causing macrophages to auto-fluoresce and undergo cell death when exposed to a certain drugs. Such transgenic mice have since played roles in investigations of macrophage activity during immune responses and macrophage activity during tumor growth.

"Prior art microinjectors function in a similar manner to macro-scale syringes: a pressure differential forces a liquid through a needle and into the cell. In some cases a glass needle that has been fire drawn from a capillary tube can be used to pierce the cellular and nuclear membranes of an oocyte. Precise pumps then cause the expulsion of minute amounts of genetic material from the needle and into the cell. Researchers have produced fine microinjection needles made from silicon nitride and silica glass that are smaller than fire drawn capillaries. These finer needles generally also employ macro-scale pumps similar to those used in traditional microinjectors.

"Pronuclear microinjection of DNA, for example, traditionally includes injection of liquid containing the DNA into the pronucleus of a cell such as a zygote. Such injections can be challenging processes due to the potential for cell lysis and chromosomal damage. In part, these challenges have motivated the development of various direct and indirect methods of transgenesis, such as viral transfection and embryonic stem cell targeting and injection. In viral transfection, a transgene is inserted into virus particles, which in turn act as carriers, delivering the genetic material to an oocyte or embryo. In embryonic stem-cell mediated transgenesis, a transgene is first targeted in vitro using a ubiquitous gene, such as ROSA, into embryonic stem cells. The transfected embryonic stem cells are then injected into blastocyst stage embryos, resulting in chimeric offspring. These chimeras must be bred to finally obtain germ line transgenic animals. In another example, existing micro-machined or carbon nanotube microelectromechanical systems (MEMS) designed for DNA delivery into tissue cultures have successfully introduced transgenes into cells, but are unsuitable for use in embryos for transgenic animal production. For example, such techniques require cells to grow around stationary needles or require extended periods of time to release bound DNA into the cells. Furthermore, such MEMS techniques do not provide sufficient mechanical displacement to penetrate a zygote's pronucleus."

In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors' summary information for this patent application: "The present disclosure provides systems, devices, and methods for delivering a biological material into an organelle of a cell. In one aspect, for example, a method for introducing biological material into an organelle of a cell can include bringing into proximity outside of a cell a lance and a preselected biological material, charging the lance with a polarity and a charge sufficient to electrically associate the preselected biological material with a tip portion of the lance, and penetrating an outer portion of the cell with the lance and directing and inserting the lance into the cell but outside of the organelle. The method can further include discharging the lance to release at least a portion of the biological material, charging the lance with an opposite polarity and charge sufficient to electrophoretically drive at least a portion of the biological material away from the lance toward the organelle, and withdrawing the lance from the cell. In one specific aspect, charging the lance with the opposite polarity and charge is sufficient to electroporate the organelle's membrane. In one aspect, charging the lance with an opposite polarity and charge is sufficient to electrophoretically move at least a portion of the biological material from the lance and into the organelle.

"In another aspect, the lance can be formed of a lance material that does not generate products toxic to the cell when the lance is charged. Additionally, the lance material can be selected to remain conductive under charged conditions. Furthermore, in some aspects the method can include bringing a counter electrode into electrical proximity of the lance to complete an electrical circuit. In one aspect, the counter electrode can be formed of an electrode material that does not generate products toxic to the cell when the lance is charged, and the electrode material remains conductive under charged conditions.

"Non-limiting examples of organelles can include a nucleus, a pronucleus, a mitochondria, a chloroplast, a vacuole, an endocytic vesicle, a lysosome, and the like. In one specific aspect, the organelle can be a pronucleus. In another specific aspect, the biological material can be simultaneously delivered into two pronuclei of the same cell.

"In another aspect, a method for transfecting a zygote with a biological material is provided. Such a method can include bringing into proximity a lance and a preselected DNA material outside of a zygote, charging the lance with a polarity and a charge sufficient to electrically associate the preselected DNA material with a tip portion of the lance, and penetrating an outer portion of the zygote with the lance and directing and inserting the lance into the cell but outside of the pronucleus. The method can further include discharging the lance to release at least a portion of the DNA material from the lance, charging the lance with an opposite polarity and charge sufficient to electrophoretically drive at least a portion of the DNA material into the pronucleus, and withdrawing the lance from the zygote.

"In a further aspect, a system for electrophoretically introducing biological material into an organelle of interest of a cell is provided. Such a system can include a lance capable of receiving and holding an electrical charge sufficient to electrostatically associate preselected biological material thereto, a charging system electrically coupleable to the lance and operable to charge and discharge the lance, and a lance manipulation system operable to move the lance into and out of the cell. The charging system can be capable of delivering an electrical charge to the lance having a voltage in excess of a decomposition voltage of the lance that is sufficient to electrophoretically transport the preselected biological material into the organelle of interest. In one specific aspect, the charging system is capable of delivering a discontinuous voltage to the lance. In another specific aspect, the charging system includes a signal generator functionally coupled to a power supply such that the signal generator gates an electrical output of the power supply to generate the discontinuous voltage. In a further specific aspect, the charging system is capable of delivering both a positive and a negative electrical charge to the lance having a voltage in excess of the decomposition voltage of the lance. In yet a further aspect, the lance has a structural configuration to allow a portion of the lance to enter the cell and be positioned in sufficient proximity to the organelle of interest to effectively delivery the preselected biological material into the organelle of interest via electrophoresis. In another specific aspect, the lance has a structural configuration to allow a portion of the lance to enter the cell and be positioned in sufficient proximity to the organelle of interest that the organelle of interest is within an electroporetic envelope of the lance when the lance is charged.

BRIEF DESCRIPTION OF THE DRAWINGS

"FIG. 1A shows a schematic representation of a step of the delivery of a biological material into a cellular organelle in accordance with one embodiment of the present disclosure.

"FIG. 1B shows a schematic representation of a step of the delivery of a biological material into a cellular organelle in accordance with one embodiment of the present disclosure.

"FIG. 1C shows a schematic representation of a step of the delivery of a biological material into a cellular organelle in accordance with one embodiment of the present disclosure.

"FIG. 1D shows a schematic representation of a step of the delivery of a biological material into a cellular organelle in accordance with one embodiment of the present disclosure.

"FIG. 1E shows a schematic representation of a step of the delivery of a biological material into a cellular organelle in accordance with one embodiment of the present disclosure.

"FIG. 1F shows a schematic representation of a step of the delivery of a biological material into a cellular organelle in accordance with one embodiment of the present disclosure.

"FIG. 2A depicts a fragment of DNA in an electric field in accordance with another embodiment of the present disclosure.

"FIG. 2B depicts a fragment of DNA in an electric field in accordance with another embodiment of the present disclosure.

"FIG. 3 shows a simulation of an electric field around a lance in accordance with another embodiment of the present disclosure.

"FIG. 4A shows a simulation of DNA movement in an electric field in accordance with another embodiment of the present disclosure.

"FIG. 4B shows a simulation of DNA movement in an electric field in accordance with another embodiment of the present disclosure.

"FIG. 5 shows a representation of pronuclear location in a cell in accordance with another embodiment of the present disclosure.

"FIG. 6 shows a schematic of an injection system in accordance with another embodiment of the present disclosure.

"FIG. 7 shows a schematic of an injection system in accordance with another embodiment of the present invention.

"FIG. 8 shows a schematic of an injection system in accordance with another embodiment of the present invention.

"FIG. 9A shows graphical representations of data in accordance with another embodiment of the present invention.

"FIG. 9B shows graphical representations of data in accordance with another embodiment of the present invention.

"FIG. 9C shows graphical representations of data in accordance with another embodiment of the present invention."

For more information, see this patent application: Aten, Quentin T.; David, Regis A.; Burnett, Sandra H.; Jensen, Brian D.; Howell, Larry L. Cytoplasma to Organelle Delivery System and Associated Methods. Filed October 18, 2013 and posted May 15, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2411&p=49&f=G&l=50&d=PG01&S1=20140508.PD.&OS=PD/20140508&RS=PD/20140508

Keywords for this news article include: Ovum, Zygote, Oocytes, Viral DNA, Organelles, DNA Research, Cellular Structures, Intracellular Space, Cytoplasmic Structures, Brigham Young University.

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Source: Life Science Weekly


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