WO2022043355A1

WO2022043355A1 - Electropulse induced mikroinjection into cells

Info

Publication number: WO2022043355A1
Application number: PCT/EP2021/073445
Authority: WO
Inventors: Boris SKRYABIN; Leonid GUBAR; Timofey ROZHDESTVENSKIY
Original assignee: Westfälische Wilhelms-Universität Münster
Priority date: 2020-08-26
Filing date: 2021-08-25
Publication date: 2022-03-03

Abstract

The present invention relates to a method of injecting a substance into a cell, comprising using a capillary that is operatively connected with an electric pulse generator and filled with a solution to be injected and applying a single electric pulse or multiple electric pulses to facilitate the penetration of the cell with said capillary, wherein the single electric pulse or multiple electric pulses are applied when penetrating the cell with said capillary. In addition, the present invention concerns a device for a microinjection according to the herein described method. Also encompassed is the use of an electric pulse generator or electric pulse for enabling the penetration of the cell according to the herein described method.

Description

ELECTROPULSE INDUCED MIKROINJECTION INTO CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority of Luxembourgish Patent Application LU 102011 filed on 26 August 2020, the content of which is hereby incorporated by reference in its entirety for all purposes.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to a method of injecting a substance into a cell, comprising using a capillary that is operatively connected with an electric pulse generator and filled with a solution to be injected and applying a single electric pulse or multiple electric pulses to facilitate the penetration of the cell with said capillary, wherein the single electric pulse or multiple electric pulses are applied when penetrating the cell with said capillary. The present invention also relates to a device for a microinjection according to the herein described method. The present invention also relates to a use of an electric pulse generator or electric pulse for facilitating penetration of a cell membrane and/or cell nucleus and/or pronuclei (pronucleus) with an injection capillary.

BACKGROUND ART

[0003] In medical and bio-pharmacological research, transgenic technologies are a powerful tool to investigate the molecular bases of diseases and to develop drugs and biopharmaceutical components for their effective treatment. For this, it is common to create transgenic animals by transferring and/or integrating genes from another species or breed. For this purpose, it is required to transfer molecular components into cells like e.g. fertilized oocytes or embryos.

[0004] There are four major methods to transfer molecular components into fertilized oocytes or embryos for generating a transgenic animal. 1) The method of passive uptake, wherein a material, loaded into liposomes-like components, is transferred to a cells in media. Thereby, the liposomes fuse with the cellular membrane thus releasing the loaded material inside the cell. Although this method allows a treatment of many cells at a time, the transfection efficiency for oocytes or embryos is extremely low. Moreover, the transfection reagents (liposomes-like components) are highly toxic for oocytes, making this method technically impractical. 2) The method of electroporation, wherein an electrical field is used to transfer material into a cell. By applying the electrical field to the cell, tiny pores are generated in the cell membrane thus increasing the penetrability for materials to diffuse into the cell. Once the electric field is released, the pores in the cellular membrane are sealed again. 3). The method of particle (components) bombardment, wherein material is ballistically transferred into a cell. This method is designed for DNA. Thus, DNA particles are bombarded towards cell membranes at high speed, which allows a penetration of cells and cellular organelles. 4). The method of mechanical penetration, wherein a cellular membrane is penetrated by a micro capillary needle using hydraulic pressure to transfer material into a cell. In a modification of this method, electroosmosis is used to transfer the material from the capillary into the cell. This allows the controlling of the fluid flow to some extend during the mechanical penetration of the cell membrane. Currently the most common method for penetrating a cell membrane is a mechanical microinjection with a micro capillary needle.

[0005] The common mechanical microinjection is material and time consuming since the needle capillary, which is often blocked by cellular components (e.g. organelles, part of membranes) during cell penetration, needs to be exchanged regularly. Further, the common mechanical microinjection has a low embryo survival rate since the person performing the experiment estimates the strength required to penetrate but not to crush a cell. This makes the common microinjection highly depending on the skill of the person performing the microinjection. Thus, there is a need for the provision of an improved method for microinjecting into a cell.

[0006] Accordingly, it is an object of the invention to provide a method of injecting a substance into a cell using a capillary that avoids these drawbacks.

SUMMARY OF THE INVENTION

[0007] This object is accomplished by the method, the device and the use of a microinjection having the features of the independent claims.

[0008] The invention provides a method of injecting a substance into a cell, comprising using a capillary that is operatively connected with an electric pulse generator and filled with a liquid to be injected and applying a single electric pulse or multiple electric pulses to facilitate the penetration of the cell with said capillary, wherein the single electric pulse or multiple electric pulses are applied when penetrating the cell with said capillary.

[0009] The invention also provides a microinjection device, comprising a capillary filled with a liquid comprising a substance to be injected, wherein the capillary is operatively connected to an electric pulse generator and wherein the electric pulse generator comprises an electronic control unit that is configured to generate one single electric pulse or multiple electric pulses to facilitate the penetration of a cell or cell membrane.

[0010] The invention further provides a use of an electric pulse generator or an electric pulse for facilitating penetration of a cell membrane and/or cell nucleus and/or pronuclei (pronucleus) with an injection capillary.

BRIEF DESCRIPTION OF THE DRAWINGS [0011] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the drawings, in which:

[0012] Figure 1 shows a schematic representation of conventional device for a microinjection. The device comprises a light microscope (1) with grounded objection table, a capillary-holding device (2), an embryo (oocyte) holding capillary (3), a microinjection capillary (4), a microsyringe (5) to produce negative hydraulic pressure for holding capillary and a device (6) to produce positive hydraulic pressure used to control solution flow inside the cell.

[0013] Figure 2 shows a schematic representation of an exemplary device suitable for carrying out a microinjection of the present invention. The device may comprise the items of a conventional microinjection device (Figure 1) and may be further equipped with an electric pulse generator (7). The pulse generator (7) may operate at 110V to 230V and may control the duration, frequency and amplitude of the electric pulse or the electric pulses. Via output cable (8), the electric pulse generator (7) may be connected to an anode (9), which may be inserted into the microinjection capillary (4). The end of the anode (9) may be contacted to the liquid inside the capillary. The objection table may be grounded (10). Additionally or alternatively, the electric pulse generator (7) may also be grounded (11).

[0014] Figure 3 shows steps of an exemplary microinjection method according to the present invention. In particular, Figure 3 schematically illustrates the use of an electric pulse for facilitating the penetration of a fertilized oocyte. The initial situation is shown in Figure 3A. The zona pellucida (1) surrounds the cell membrane (2) of a fertilized oocyte containing two fusing pronuclei (3). The injection needle (4) is directed straight at the zona pellucida (1) of a cell (oocyte), which is located in front of the holding capillary (5) and which is to be penetrated. Figure 3B shows the mechanical penetration of the zona pellucida (1) by the injection needle (4). Thereby, the injection needle (4) is positioned directly in front of the oocyte to be penetrated. In Figure 3C the cell penetration of the present invention is shown. The injection needle (4) is carefully pushed towards the oocyte membrane forming a small bulge (6). At this particular moment, an electrical pulse or more electrical pulses are applied facilitating the penetration of the oocyte. After penetrating, the injection solution (7) is injected into the penetrated oocyte. This process is shown in Figure 3D.

[0015] Figure 4 shows another schematic representation of an exemplary device suitable for carrying out a microinjection of the present invention. The device may comprise the items of the device for a microinjection shown in Figure 2 but with a pulse generator (7) that has an output cable (12) connected to a cathode, which may be connected to the holding capillary (2). The cathode may also be fixed on a dielectric attached to the device for the holding capillary (not shown in the drawing).

[0016] Figure 5 shows a schematic representation of pulse shapes in a diagram where time is plotted against voltage. These pulses are inter alia applicable in the microinjection of the present invention. Figure 5a depicts a triangle shaped pulse with an immediate rising and a slow falling voltage. Figure 5b depicts three triangle shaped pulses with an immediate rising and a slow falling voltage each forming a sawtooth shaped pulse. Figure 5c depicts an L-shaped pulse. This pulse starts with a pulse package of multiple electric pulses with high amplitudes followed by a single pulse with a prolong duration and low amplitude.

[0017] Figure 6 shows a schematic representation of pulse shapes and pulse packages in diagrams where time is plotted against voltage. These pulse shapes and pulse packages are inter alia applicable to facilitate the cell penetration of the present invention. ‘P_on’ refers to the duration time of a pulse package (pulse package width). ‘T_on’ refers to the duration time of a single pulse (pulse width) and ‘T_Off’ refers to the time without a pulse. Figure 6a illustrates the course of a direct current (DC). Figure 6b depicts a single electric pulse. Figure 6c depicts multiple electric pulses in a single electric pulse package. Figure 6d depicts multiple electric pulses in 4 electric pulse packages consecutively applied (drilling). Figure 6e depicts 4 single electric pulses consecutively applied (drilling).

[0018] Figure 7: Panels A and B show the percentage of survival of C57BL6/J embryos after electropulse induces microinjections (EPI-MI, n=156) and conventional mechanical microinjections (Meeh. -Ml, n=192). Panels C and D show the percentage of survival of B6D2F1 embryos after electropulse induced microinjections (EPI-MI, n=128) and conventional mechanical microinjections (Mech.-MI, n=131 ).

DETAILED DESCRIPTION OF THE INVENTION

[0019] It has been surprisingly found by the inventors of the present application that using an electric pulse or multiple electric pulses as described herein facilitates cell penetration for microinjection. Consequently, the penetration of the cell with the capillary may be facilitated. The methods and devices of the present invention may also prevent the blocking of the capillary with cellular components or cell membrane residues during the penetration. Consequently, the present invention may reduce the frequency of a manual exchange of the blocked capillary. The present invention may also increase the embryo survival rate since the electro pulse facilitated cell penetration may reduce the force effects of the capillary on the surface of the cell to be penetrated. Thereby, the method of the present invention is less prone to errors. Thus, the present invention provides an easy and material and time saving microinjection method.

[0020] As explained above, the invention relates to a method of injecting a substance into a cell, comprising using a capillary that is operatively connected with an electric pulse generator and filled with a solution to be injected and applying a single electric pulse or multiple electric pulses to said capillary, wherein the single electric pulse or multiple electric pulses are applied when penetrating the cell with said capillary. It is thus envisioned that said single electric pulse or multiple electric pulses are applied concurrently with the penetration. Said single electric pulse or multiple electric pulses is considered to facilitate the penetration. Accordingly, penetration is preferably effected by a combination of mechanical stress directed to the cell by the capillary and the application of said single electric pulse or multiple electric pulses.

[0021] When used herein, the term ‘injecting’ comprises any introduction of a substance, such as a liquid which may comprise a substance, into a cell by the use of a hollow means allowing passing through the substance into the cell. The hollow means may be a capillary. In the present invention, the hollow means may be a capillary suitable for microinjections, such as a needle capillary. To inject a cell, the cell is typically penetrated by the hollow means. Once the cell is penetrated by the hollow means, injection of the substance is preferably conducted mechanically.

[0022] In the present invention, an electric pulse or multiple electric pulses may be applied to a capillary to facilitate a penetration for microinjection into a cell. Therefore, any electric pulse(s) suitable to facilitate the penetration for microinjection into a cell with a capillary can be applied. Consequently, the electric pulse(s) may be modulated by its/their parameters, which are duration time, frequency, and amplitude. For example, the electric pulse or multiple electric pulses suitable for the present invention may have at least a duration time of 0.1ms. The duration time of the electric pulse or multiple electric pulses may also be in the range of about 0.1ms to about 3ms, such as from bout 0.2ms to about 2ms, such as from about 0.3ms to about 1ms. In one example, the duration time of the electric pulse or multiple electric pulses may be about 0.4ms to about 0.8ms. Multiple electric pulses can form a pulse package as shown in Fig. 6. If multiple electric pulses are applied, the frequency of the pulses may also be modulated. The electric pulses suitable for the present invention may have a frequency of at least 1Hz. Thus, the electric pulses may also have a frequency from about 1 Hz to about 400kHz. In one example, the multiple electric pulses have a frequency of about about 10 kHz to about 400 kHz, such as about 15 kHz to about 300 kHz, about 20 kHz to about 250 kHz, about 25 kHz to about 200 kHz, about 30 kHz to about 150 kHz, about 35 kHz to about 120 kHz. In one example, the multiple electric pulses have a frequency of about 40kHz to about 100kHz.

[0023] The electric pulse or multiple electric pulses may have an amplitude of at least 0.5V. In one example, an electric pulse or multiple electric pulses may have an amplitude from about 0.5V to about 400V. For example, an electric pulse or multiple electric pulses may have amplitude from about 10 V to about 400V, such as about 15 V to about 300V, about 20 V to about 250V, about 25V to about 200V, about 30V to about 150V, about 35V to about 120V, about 40V to about 100V, or about 100V.

[0024] The parameters amplitude, duration time of either a single pulse (if no pulse package is applied) or a pulse package, and number of single pulses or pulse packages may be expressed in form of the following formula:

E*P_w*n= x wherein:

E is the amplitude in Volts, Pw is duration time of a single pulse or a pulse package in seconds, n is number of single pulses or pulse packages (n>1).

According to the present disclosure, the value for x may be in a range from about 10'⁴ V*s to about 50V*s. Preferably, the value for x may be in a range of about 0.1 to about 30 V*s. Preferably, the value for x may be in a range of about 0.2 to about 25 V*s. Preferably, the value for x may be in a range of about 0.3 to about 20 V*s. Preferably, the value for x may be in a range of about 0.5 to about 15 V*s. Preferably, the value for x may be in a range of about 1 to about 10 V*s. Preferably, the value for x may be in a range of about 1 to about 5 V*s. Preferably, the value for x may be in a range of about 2 to about 4 V*s. Preferably, the value for x may be about 3 V*s.

[0025] Turning to the parameter amplitude, an electric pulse(s) revealing an immediate raise of amplitude may be preferred for the present invention. In this regard, the electric pulse or multiple electric pulses being applied to a capillary to facilitate a penetration for microinjection into a cell may have an immediate raise in amplitude. The immediate raise in amplitude may be described, for example, wherein the maximum of the amplitude is reached within a half, preferably a third, preferably a fourth, preferably a fifth, or preferably a tenth of the duration of the pulse. In this regard, the electric pulse or multiple electric pulses may, for example, be triangle shaped when time is plotted against voltage as for example shown in Figure 5a. In one example, electric pulse or multiple electric pulses may be sawtooth-shaped or L-shaped when time is plotted against voltage as for example in Figure 5b. In a preferred example, the pulse(s) may be rectangular shaped, when time is plotted against voltage as for example in Figure 5c.

[0026] In the present invention, any pulsating current suitable for generating the electric pulse or multiple electric pulses may be applied to facilitate a cell penetration for microinjection. In one example of the present invention, a pulsating direct current (DC) may be applied to generate the electric pulse or multiple electric pulses required for the cell penetration.

[0027] To further optimize the method of the present invention, the electric pulse or electric pulses may also be modulated by two parameters. An example of this is an electric pulse or are multiple electric pulses being modulated by frequency and duration time of the pulse. Accordingly, it may be that the pulse frequency is set high and the duration time is set low resulting in many pulses with a short duration time. In this regard, it may also be that the frequency is set low and the duration time is set high resulting in only a few pulses with a long duration time. In one example, the frequency and the duration time is set high resulting in many pulses with a long duration time. In one example the frequency and the duration time is set low resulting in a few pulses with a short duration time.

[0028] As mentioned above, an electric pulse or multiple electric pulses are used in the present invention to facilitate a cell penetration for microinjection into the cell. Accordingly, one example of the present invention may comprise the application of a single electric pulse to facilitate the penetration for microinjection (Figure 6b). The single electric pulse may be characterized by the parameters amplitude and duration time.

[0029] According to the present invention, multiple electric pulses may be applied. These multiple pulses can be in form of a series of single pulses, in form of a single pulse package, or in form a a series of multiple pulse packages. A pulse package may consist of multiple electric pulses within a constant duration time. In one example of the present invention, multiple electric pulses may be applied in a single pulse package (Figure 6c). Multiple electric pulses may also be applied in multiple pulse packages, which may be applied consecutively (Figure 6d). Multiple electric pulse packages may also consist of a series of single pulses (Figure 6e). The consecutive application of multiple pulse packages consisting of multiple electric pulses may be termed ‘drilling’. The multiple electric pulses of the pulse package or pulse packages may be characterized by the parameters duration time of a single pulse (T on), duration time of the pulse package (P on), Time of drilling (T drilling), package period, pulse period, package frequency, pulse frequency, and/or amplitude. When applying the present invention the parameters may be modulated when applying the method, e.g. within one drilling. This means that one or more of the aforementioned parameters may change during the application of the method. It is however also possible that one or more, or even all parameters are set constant when applying the method. Thus, in one example, the multiple electric pulses may have a constant voltage. In this context, the constant voltage refers to the amplitude of the electric pulse or multiple electric pulses. Thus, the consecutively applied multiple electric pulses of the pulse package or the multiple pulse packages (drilling) may also have a constant voltage. Further, the pulse packages consisting of single electric pulses (drilling) may also have a constant voltage. Further, it may be that the pulse package or pulse packages (drilling) may consist of multiple electric pulses with a constant frequency. It also may be that the pulse packages (drilling) may consist of single electric pulses with a constant frequency. In one example, pulse package or pulse packages (drilling) may consist of multiple electric pulses with a constant duration time. In another example, the pulse packages (drilling) may consist of single electric pulses with a constant duration time. Consequently, a pulse package consisting of a single electric pulse (Figure 6b) or multiple electric pulses (Figure 6c) may have a duration time of at least about 100 pSec. In one example of the present invention, the pulse package consisting of a single electric pulse or multiple electric pulses may have a duration time of about 100 pSec to about 3000 pSec. Thus, in one example, the pulse package consisting of a single electric pulse or multiple electric pulses may have a duration time of about 150 pSec to about 2500 pSec, about 200 pSec to about 2000 pSec, about 250 pSec to about 1500 pSec, about 300 pSec to about 1200 pSec, about 350 pSec to about 1000 pSec, about 400 pSec to about 900 pSec, about 450 pSec to about 800 pSec, about 500 pSec to about 750 pSec, about 550 pSec to about 700 pSec, or about 600 pSec. In this context, multiple pulse packages consecutively applied (drilling) and consisting of multiple pulses (Figure 6d) or single pulses (Figure 6e) or may also have a constant duration time. In one example of the present invention, the constant duration time of the multiple pulse packages may be at least about 100 pSec. In one example, the constant duration time of the multiple pulse packages may be about 150 pSec to about 2500 pSec, about 200 pSec to about 2000 pSec, about 250 pSec to about 1500 pSec, about 300 pSec to about 1200 pSec, about 350 pSec to about 1000 pSec, about 400 pSec to about 900 pSec, about 450 pSec to about 800 pSec, about 500 pSec to about 750 pSec, about 550 pSec to about 700 pSec, or about 600 pSec.

[0030] In preferred embodiments, a single pulse package (Fig.6C) is applied with a duration time of 600 pSec, with an amplitude range between 40 - 100V and frequency range between 40kHz to 100kHz. In preferred embodiments, multiple packages (Fig 6d) are applied, wherein the multiple pulse packages consist of about 20 to about 50 single pulse packages of 600 pSec each in one multiple package, wherein the multiple package duration is preferably about 1sec. The amplitude and frequency ranges of the multiple packages are preferably in the range of 40- 100V and 40-100kHz.

[0031] A microinjection may be performed with a capillary, which can penetrate a cell and thus allowing the microinjection of a substance into the cell. For cell penetration before microinjection, the capillary may be directed straight towards the cell to be penetrated for microinjection. In the present invention, any capillary suitable for microinjecting a cell can be used. An illustrative example for such a capillary may be a microinjection needle. During the microinjection of the present invention, a single electric pulse or multiple electric pulses or packages thereof may be applied to the capillary facilitating the penetration for microinjection into the cell. For electric pulse transmission, the capillary may be operatively connected to an electrode. This means that the capillary is connected to the electrode, which may be a cathode or an anode, in a way that one electric pulse or multiple electric pulses can be conducted from the electrode to the capillary. Further, the electrode being connected to the capillary may be connected to a device suitable to generate one or more electric pulses. An illustrative example for such a device may be an electric pulse generator. In one example, the capillary may be connected to a cathode, which may be connected to an electric pulse generator. In a preferred example, the capillary may be connected to an anode, which may be connected to an electric pulse generator. The electrodes being connected to the capillary may be placed inside the capillary. In one example, an anode is placed inside the capillary. Either way, the electrodes may be made from an electro-conductive material. In the present invention, any material suitable to conduct electrons to the capillary can be used. An example for such an electro- conductive material is a metal such as silver, copper, gold, aluminum, brass, zinc, nickel, phosphor bronze, iron, platinum, tin, chrome, titanium, stainless steel or combinations thereof. Preferably, the electrode for use in the microinjection of the present invention is of a nobel metal, such as gold or platinum, with platinum being preferred. Thus, in one example, an anode placed inside the capillary is made of platinum. In contrast, the capillary may be made of a solid electro unconducive material such as plastic or glass, such as silica glass.

[0032] Before the microinjection of the present invention may be performed, the capillary may be filled with a liquid comprising a substance suitable to be injected into a cell. The liquid may herein also be termed injection solution. The injection solution may comprise an aqueous solution, a physiological solution, or a salt solution. In the present invention, the injection solution may preferably comprise a physiological solution or a salt solution. An example of a physiological solution suitable for the present invention is a sodium chloride solution, a Tris-HCL buffer optionally comprising EDTA, or a Yamamoto Ringer solution. A salt solution suitable for the present invention may be prepared with any conventional salt suitable for generating a physiological salt solution. An example for such a salt is sodium chloride, potassium chloride, potassium acetate or potassium iodate. As an illustrative example, a 0.6 mM Hepes (pH=7.5), 2 mM potassium acetate microinjection buffer may be used as described in Skryabin et al., 2019 (https://doi.org/10.1101/570739). In the present invention, the salt concentration of the solution may be at least 0.1 pM. In one example, the salt concentration may be in a range about 0.1 pM to 5M. Thus, in one example, the salt concentration may be about 0.1 mM to about 150mM, about 0.2mM to about 100mM, about 0.5 mM to about 50mM, preferably 1 to 10 mM, preferably 1.5 to 5 mM. As an illustrative example, an injection solution may comprise about 0.6 mM Hepes and/or about 2 mM potassium acetate.

[0033] According to the present invention, a capillary used for cell penetration is preferably directed towards the cell surface. The cell is preferably penetrated while applying an electric pulse or multiple electric pulses or packages thereof (drilling) on the capillary. In this context, cell penetration may comprise penetration of a cell membrane or a zona pellucida. It is also possible to penetrate a cell compartment, such as a nucleus (pronucleus), an intercisternal space, the cytoplasm, a cell organelle or a membrane of such an intracellular structure.

[0034] The purpose of the microinjection of the present invention may be to inject a substance into a cell by the means of a capillary. Here, a solution may function as a carrier for a substance or material to be injected into cell. In the present invention, any substance or material suitable for being injected into a cell can be used. An example for a substance or material being microinjected may be an entire cell such as a prokaryotic or eukaryotic cell or parts thereof, wherein the parts of such a cell may comprise a cell compartment such as a cell organelle. In one example, a cell membrane, a cytoplasm, a nucleus, a Golgi apparatus, an endoplasmic reticulum, a mitochondrion, a lysosome, or pieces or combinations thereof may be microinjected into a cell. The substance or material to be microinjected may also be a virus or a virus particle or a biological molecule such as a polymer, a peptide, or a protein, such as an enzyme. Molecules such as a nanoparticle(s) (made of any chemical element or substance) or an amino acid may also be injected into a cell. In some preferred embodiments, genetic material may be the substance to be injected according to the present invention. Such genetic material may be a substance selected from a group consisting of deoxyribonucleotides, ribonucleotides, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), such as a double stranded deoxyribonucleic acid (dsDNA), a single stranded deoxyribonucleic acid (ssDNA), a double stranded ribonucleic acid (dsRNA), a single stranded ribonucleic acid (ssRNA), oligodeoxyribonucleotide(s) (ODN), oligoribonucleotide(s) (ORN), transcription factors, and combinations thereof. Preferred substances are DNA and/or RNA. Additionally, a substance may be a chemical such as a drug or a chemically modified amino acid or a chemically modified nucleic acid, which comprises modifications within the polymeric chain of the dsDNA, ssDNA, ODN, dsRNA, ssRNA, ORN. Also, combinations of the herein listed examples may be used as substances according to the present invention. In one example of the present invention, a pronuclear injection may be performed. For this procedure, a DNA fragment may be microinjected into a pronucleus (pronuclei) of the cell. Accordingly, the present invention may be suitable for the generation of a transgenic cell, a transgenic organism, such as a transgenic bacterium, a transgenic fungus, a transgenic plant, or a transgenic animal. The present invention may be suitable for in vitro fertilized oocytes (zygotes).

[0035] The microinjection of the present invention can be performed on any cell suitable for being injected. An example for such a cell may be a prokaryotic cell or a eukaryotic cell. A suitable eukaryotic cell for the present invention may be any cell of the protista kingdom, the plant kingdom, the fungus kingdom or the animal kingdom. An example for a plant cell suitable for the present invention may also be a protoplast. An example for an animal cell suitable for the present invention may be a cell of a vertebrate or a cell of an invertebrate. An example for an invertebrate is an arthropod, such as an insect, such as a fly, an arachnid such as a spider, a mollusk such as a snail, an annelid such as a worm, a crustacean such as a crab or a cnidarian such as a coral. An example for a vertebrate is a fish such as a medaka or zebrafish, a bird such as a chicken or a turkey, a reptile such as a lizard or a snake, an amphibian such as a frog, or a mammal. In a preferred example, the cell suitable for the microinjection of the present invention may be a mammalian cell. The mammalian cell may comprise a cell of a human or a non-human animal. For example, the animal cell suitable for the present invention may be from a mouse, a rat, a rabbit, a primate, a camellia, a pig, a cow, a horse, a donkey, a cat, a dog, a sheep, a goat, a guinea pig, a hamster or a human. In the present invention, the cell suitable for microinjection may also be a specific cell such as a myeloid cell, a lymphatic cell or a germ cell. The cell suitable for microinjection may be an oocyte, a fertilized oocyte (zygote), or an embryonic cell. In one example, the present invention may be performed on an embryo comprising an embryo in a one cell stage and an embryo in a multiple cell stage. The multicellular stage of an embryo may comprise a 2-cell stage, a 4-cell stage, or an 8-cell stage. However, a human embryonic stem cell or a human germ line cell may be excluded from the present invention. [0036] If the present invention is performed on a fertilized oocyte (zygote), the microinjection may be performed within a few hours (h) after fertilization, since the fertilization may cause an increase of viscosity within the cytoplasm of the zygote. For example, the microinjection of the present invention may be performed between Oh to about 11 h after fertilization. Further, the number of electric pulses or pulse packages may have to be selected depending on the cytoplasm viscosity. For example, a microinjection performed shortly after fertilization may require less electric pulses or pulse packages in comparison to a microinjection performed hours after fertilization. In one example of the present invention, multiple electric pulses may be applied to a zygote about 5h to 7h after fertilization. In one example, a single electric pulse may be sufficient to penetrate a zygote about 5h or about 6h after fertilization.

[0037] For implementing the herein described microinjection, the present invention further envisions any microinjection device suitable for injecting a cell by the use of a single electric pulse or multiple electric pulses to facilitate the penetration into the cell. Thus, the microinjection device of the present invention may comprise a device configured to generate a single electric pulse or multiple electric pulses or packages thereof. An example of such a device is an electric pulse generator that may comprise an electronic control unit that may be configured to generate one single electric pulse or multiple electric pulses. The microinjection device suitable for the present invention may further comprise a capillary, which may be filled with a liquid comprising a substance to be injected. Consequently, in one example, the device may comprise a capillary, which may be operatively connected to an electric pulse generator; wherein the electric pulse generator may comprise an electronic control unit that may be configured to generate one single electric pulse or multiple electric pulses to facilitate the electro pulse applied penetration in a cell or cell membrane.

[0038] In the present invention, the electric pulse generator may be configured to generate an electric pulse or multiple electric pulses suitable for cell penetration. The electric pulse generator may be configured to generate an electric pulse or multiple electric pulses as described for any of the methods of the disclosure. The electric pulse or multiple electric pulses may be characterized by the parameters duration time of a single pulse (T on), duration time of the pulse package (P on), Time of drilling (T drilling), package period, pulse period, package frequency, pulse frequency, and/or amplitude. In one example, the electric pulse generator may be configured to generate an electric pulse or multiple electric pulses that may have a duration time of at least 100 pSec. In one example, the electric pulse generator may be configured to generate an electric pulse or multiple electric pulses that may have a duration time in the range of about of about 100 pSec to about 3000 pSec, such as from about 150 pSec to about 2500 pSec, about 200 pSec to about 2000 pSec, about 250 pSec to about 1500 pSec, about 300 pSec to about 1200 pSec, about 350 pSec to about 1000 pSec, about 400 pSec to about 900 pSec, about 450 pSec to about 800 pSec, about 500 pSec to about 750 pSec, about 550 pSec to about 700 pSec, or about 600 pSec. In a further example, the electric pulse generator may be configured to generate an electric pulse or multiple electric pulses that may have a frequency of at least 1Hz. Thus, the electric pulse generator may also be configured to generate an electric pulse or multiple electric pulses may have a frequency from about 1 Hz to about 400kHz. In one example, the electric pulse generator may be configured to generate an electric pulse or multiple electric pulses that may have a frequency of about 10 kHz to about 400 kHz, such as about 15 kHz to about 300 kHz, about 20 kHz to about 250 kHz, about 25 kHz to about 200 kHz, about 30 kHz to about 150 kHz, about 35 kHz to about 120 kHz. In one example, the multiple electric pulses have a frequency of about 40kHz to about 100kHz. Further, it may be that the electric pulse generator may be configured to generate an electric pulse or multiple electric pulses that may have an amplitude of at least 0.5V. In one example, the electric pulse generator may be configured to generate an electric pulse or multiple electric pulses that may has an amplitude from about 0.5V to about 400V. Thus, in one example, the electric pulse generator may be configured to generate an electric pulse or multiple electric pulses that may have amplitude from about 10 V to about 400V, such as about 15 V to about 300V, about 20 V to about 250V, about 25V to about 200V, about 30V to about 150V, about 35V to about 120V, about 40V to about 100V, or about 100V.

[0039] Turning to the parameter amplitude, the electric pulse generator may be configured to generate any electric pulse or multiple electric pulses revealing an immediate raise of amplitude, which may be suitable for the present invention. In this regard, the electric pulse generator may be configured to generate an electric pulse or multiple electric pulses that may have a shape with an immediate raise in amplitude. For example, the electric pulse generator may be configured to generate an electric pulse or multiple electric pulses that may be triangle shaped when time is plotted against voltage. In one example, the electric pulse generator may be configured to generate an electric pulse or multiple electric pulses that may be sawtoothshaped or L-shaped when time is plotted against voltage. The electric pulse generator may be configured to further generate an electric pulse or multiple electric pulses that may be squareshaped, when time is plotted against voltage. In a preferred example, the electric pulse generator may be configured to generate an electric pulse or multiple electric pulses that may be rectangular-shaped, when time is plotted against voltage.

[0040] The electric pulse generator may be configured to generate a pulsating direct current (DC) to generate an electric pulse or multiple electric pulses suitable to facilitate cell penetration for microinjection.

[0041] The electric pulse generator may be configured to generate a single electric pulse or multiple electric pulses. The electric pulse generator may also be configured to generate a single pulse package consisting of multiple electric pulses. In one example, the electric pulse generator may be configured to generate a pulse package consisting of single pulses applied consecutively. In another example, the electric pulse generator may be configured to generate a pulse package consisting of multiple pulses applied consecutively. The electric pulse generator may be configured to generate an electric pulse or multiple electric pulses that may have a constant voltage. Consequently, the electric pulse generator, for example, may also be configured to generate single consecutively applied electric pulses (drilling), which may have a constant voltage. In a further example, the electric pulse generator may be configured to generate multiple pulse packages consisting of multiple electric pulses (drilling) that may have a constant voltage. In one example, the electric pulse generator may be configured to generate an electric pulse or multiple electric pulses that may have a constant frequency. Consequently, the electric pulse generator may also be configured to generate single consecutively applied electric pulses that may have a constant frequency. Further, the electric pulse generator may also be configured to generate a single or multiple pulse packages consisting of multiple electric pulses, which may have a constant pulse frequency. In a further example, the electric pulse generator may be configured to generate an electric pulse or multiple electric pulses that may have a constant duration time. Thus, the electric pulse generator may be configured to generate single consecutively applied electric pulses that may have a constant duration time. Further, the electric pulse generator may also be configured to generate a single or multiple pulse packages consisting of multiple electric pulses, which may have a constant duration time. Consequently, the electric pulse generator may be configured to generate a pulse package consisting of a single electric pulse or multiple electric pulses that may have a duration time of at least about 100 pSec. In one example of the present invention, the electric pulse generator may be configured to generate a pulse package consisting of a single electric pulse or multiple electric pulses that may have a duration time of about 100 pSec to about 3000 pSec. Thus, in one example, the electric pulse generator may be configured to generate a pulse package consisting of a single electric pulse or multiple electric pulses that may have a duration time of about 150 pSec to about 2500 pSec, about 200 pSec to about 2000 pSec, about 250 pSec to about 1500 pSec, about 300 pSec to about 1200 pSec, about 350 pSec to about 1000 pSec, about 400 pSec to about 900 pSec, about 450 pSec to about 800 pSec, about 500 pSec to about 750 pSec, about 550 pSec to about 700 pSec, or about 600 pSec. In this context, the electric pulse generator may also be configured to generate multiple pulse packages consecutively applied (drilling) and consisting of single pulses or multiple pulses, which may also have a constant duration time. In one example of the present invention, the electric pulse generator may be configured to generate multiple pulse packages, which may have a constant duration time of at least about 100 pSec. In one example, the electric pulse generator may be configured to generate multiple pulse packages that may have a constant duration time of about 100 pSec to about 3000 pSec. Thus, in one example, the electric pulse generator may be configured to generate multiple pulse packages that may have a constant duration time about about 150 pSec to about 2500 pSec, about 200 pSec to about 2000 pSec, about 250 pSec to about 1500 pSec, about 300 pSec to about 1200 pSec, about 350 pSec to about 1000 pSec, about 400 pSec to about 900 pSec, about 450 pSec to about 800 pSec, about 500 pSec to about 750 pSec, about 550 pSec to about 700 pSec, or about 600 pSec.

[0042] The device of the present invention may comprise an element, which may contribute to keeping the cell to be penetrated in place during microinjection. For this purpose, any element suitable to hold but not squeeze or damage the cell to be penetrated can be used. An example for such an element is a holding capillary. Thus, the device of the present invention may comprise a holding capillary. The holding capillary may further be connected to an electrode. Therefore, a holding capillary may be connected to an anode or a cathode. In a preferred example, a device of the present invention may comprise a holding capillary, which is connected to a cathode of the electric pulse generator. However, instead of being connected to a holding capillary, the electric pulse generator may also be grounded. Further preferred elements of the device of the present invention are shown in Figures 2 and 4. Accordingly, the device may comprise a light microscope (1 ), an objection table that may be grounded (10), a capillaryholding device (2) for a microinjection capillary (4), a microinjection capillary (4), a capillaryholding device (2) for a holding capillary (2), a holding capillary (3), a micro-syringe (5) which can be used to produce negative hydraulic pressure for a holding capillary and a device (6) which can be used to produce positive hydraulic pressure and which can be used to control solution flow inside the cell, an electric pulse generator (7), which may operate at 110V to 230V and may control the duration, frequency and amplitude of the electric pulse or the electric pulses, an output cable (8) connecting the electric pulse generator (7) to an anode (9), and/or an anode (9) which may be inserted into the microinjection capillary (4).

[0043] The present invention further envisions use of an electric pulse generator, which may be configured to generate a single electric pulse or multiple electric pulses or packages thereof, for facilitating penetration into a cell with a capillary. The electric pulse generator may be comprised in a device according to the disclosure. The electric pulse generator may be configured to generate one or more electric pulse(s) according to the disclosure. The present invention further comprises the use of a single electric pulse or multiple electric pulses or packages thereof to facilitate the penetration into a cell membrane, a pronuclear cell or a cell compartment for microinjection. An example for such a cell compartment may be a nucleus (pronucleus), an intercisternal space, the cytoplasm, a cell organelle, or a membrane of such an intracellular structure.

[0044] The present invention is further characterized by following items:

1 . A method of injecting a substance into a cell, comprising: using a capillary that is operatively connected with an electric pulse generator and filled with a liquid to be injected; and applying a single electric pulse or multiple electric pulses to said capillary; wherein the single electric pulse or multiple electric pulses are applied when penetrating the cell with said capillary. The method of item 1, wherein the electric pulse(s) has/have a duration time from about 100 pSec to about 3000 pSec, preferably about 400 pSec to about 900 pSec. The method of item 1 or 2, wherein the electric pulse(s) has/have a frequency from about 1Hz to about 400kHz, preferably about 40kHz to about 100kHz. The method of any one of items 1 to 3, wherein the electric pulse(s) has/have an amplitude from about 0.5V to about 400V, preferably about 40V to about 100V. The method of any one of items 1 to 4, wherein the relation of the parameters amplitude, pulse duration and number of pulses is as follows:

E*Pw*n= x wherein:

E is the amplitude in Volts;

Pw is the duration time of a single pulse or a pulse package in seconds; n is number of single pulses or pulse packages (n>1), wherein x has a value of at least about 10'⁴ V*s, to about 50V*s. The method of any one of items 1 to 5, wherein the electric pulse(s) has/have an immediate raise of amplitude. The method of any one of items 1 to 6, wherein the electric pulse(s) is/are rectangularshaped, square-shaped, triangle-shaped, sawtooth-shaped or L-shaped. The method of any one of items 1 to 7, wherein the electric pulse(s) is/are rectangularshaped. The method of any one of items 1 to 8, wherein a pulsating direct current (DC) is applied. The method of any one of items 1 to 9, wherein the electric pulse(s) is/are modulated by frequency and time. The method of any one of items 1 to 10, wherein a single pulse is applied. The method of any one of items 1 to 10, wherein multiple electric pulses in a single pulse package are applied. The method of any one of items 1 to 10, wherein multiple electric pulse packages are applied consisting of multiple electric pulses. The method of any one of items 1 to 10, wherein multiple electric pulse packages are applied consisting of single pulses. The method of item 11 , wherein a single electric pulse has a constant voltage. The method of item 12, wherein multiple electric pulses have a constant voltage. The method of item 13, wherein the multiple electric pulses of the pulse package or multiple pulse packages have a constant voltage. The method of item 14, wherein the pulse packages consist of single electric pulses have a constant voltage. The method of any one of items 1 to 19, wherein the single electric pulse, multiple electric pulses, single electric pulse packages or multiple pulse packages have a constant pulse frequency. The method of any one of items 10 to 19, wherein the single electric pulse or the multiple electric pulses of a pulse package or multiple pulse packages has/have a constant pulse duration time. The method of any one of items 10 to 20, wherein the single electric pulse or the multiple electric pulses of a pulse package or multiple pulse packages has/have a duration time of about 100 pSec to about 3000 pSec, preferably about 400 pSec to about 900 pSec. The method of any one of items12 to 18, wherein the single or multiple electric pulse packages consists of single pulses or multiple pulses having a duration time of about 100 pSec to about 3000 pSec, preferably about 400 pSec to about 900 pSec. The method of any one of items 1 to 22, wherein the capillary is directed towards the cell to be penetrated. The method of any one of items 1 to 23, wherein the capillary is operatively connected to an anode that is connected to an electric pulse generator. The method of item 24, wherein the anode is placed inside the capillary. The method of any one of items 23 to 25, wherein the anode is an electrode that is made of an electro-conductive material, preferably platinum. The method of any one of items 1 to 26, wherein the capillary is of an electro-unconductive material, preferably silica glass. The method of any one of items 1 to 27, wherein the capillary is filled with a salt injection solution. The method of item 28, wherein the injection solution has a salt concentration of at least 0.1 pM to about 5M, preferably about 0.5mM to about 10mM. The method of any one of items 1 to 29, wherein the method comprises penetration of a cell membrane, penetration of a zona pellucida, penetration of a cell compartment or penetration of membranes of an intracellular structure. The method of any one of items 1 to 30, wherein the method comprises injection of a deoxyribonucleotide(s), ribonucleotide(s), a deoxyribonucleic acid (DNA), ribonucleic acid (RNA), double stranded deoxyribonucleic acid (dsDNA), single stranded deoxyribonucleic acid (ssDNA), double stranded ribonucleic acid (dsRNA), single stranded ribonucleic acid (ssRNA), oligodeoxyribonucleotide(s) (ODN), oligoribonucleotide(s) (ORN), chemically modified nucleic acid(s) (including those modifications within polymeric chain of dsDNA, ssDNA, ODN, dsRNA, ssRNA, ORN), amino acid(s), a peptide, a polypeptide, a protein, chemically modified amino acid(s) (including those within a peptide, a polypeptide, a protein), an enzyme, a drug, a small molecule, nanoparticles, a transcription factor, larger structures, such as a prokaryotic or eukaryotic cells, sperm, different organelles including nucleus and mitochondria, a virus, a virus partials, or a combination thereof. The method of any one of items 1 to 31, wherein the injection comprises pronuclear injection. The method of any one of items 1 to 32, wherein the cell is a eukaryotic cell. The method of any one of items 1 to 33, wherein the cell is a protoplast, a plant cell, a fungus cell or an animal cell. The method of any one of items 1 to 34, wherein the cell is a vertebrata cell including a fish cell, an amphibians cell, a reptiles cell, a birds cell, a mammalians cell, or Invertebrata cell including an arthropods cell, a mollusks cell, an annelids cell and cnidarians cell. The method of any one of items 1 to 35, wherein the cell is a human cell, a mouse cell, a rat cell, a rabbit cell, a non-human primate cell, a pig cell, a camellia cell, a cow cell, a horse cell, a goat cell, a cat cell, a dog cell or a sheep cell. The method of any one of items 1 to 36, wherein the cell comprises an oocyte, a fertilized oocyte or an embryotic cell. The method of any one of items 1 to 37, wherein the cell is a cell of an embryo in one cell stage or zygote. The method of any one of items 1 to 38, wherein the cell is a cell of an embryo in multicellular stage. The method of any one of items 1 to 39, wherein the cell is not a human embryonic stem cell or a human germ line cell. The method of any one of items 1 to 40, wherein the cell is a fertilized oocyte between Oh to about 11 h after fertilization, preferably 5h to about 7h after fertilization, and wherein multiple electric pulses are applied to the cell. The method of any one of items 1 to 41 , wherein the cell is a fertilized oocyte about 5h or more, preferably about 6h or more after fertilization and wherein a single electric pulse or multiple electric pulses is/are applied to the cell. The method of any one of items 1 to 42, wherein the single electric pulse or multiple electric pulses are applied concurrently with penetrating the cell. The method of any one of items 1 to 43, wherein the substance is injected by essentially mechanical means, preferably mechanically. The method of any one of items 1 to 44, wherein penetration is effected by a combination of mechanical stress directed to the cell by the capillary and application of said single electric pulse or multiple electric pulses. A microinjection device, comprising a capillary filled with a liquid comprising a substance to be injected; wherein the capillary is operatively connected to an electric pulse generator; and wherein the electric pulse generator comprises an electronic control unit that is configured to generate one single electric pulse or multiple electric pulses to facilitate the penetration of a cell or cell membrane. The device of item 46, wherein the electric pulse generator is configured to generate one or more electric pulse(s) that has/have a duration time from about 100 pSec to about 3000 pSec, preferably about 400 pSec to about 900 pSec. The device of item 46 or 47, wherein the electric pulse generator is configured to generate one or more electric pulse(s) that has/have a frequency from about 1 Hz to about 400kHz, preferably about 40kHz to about 100kHz. The device of any one of items 46 to 48, wherein the electric pulse generator is configured to generate one or more electric pulse(s) that has/have a/an amplitude from about 0.5V to about 400V, preferably about 40V to about 100V. The device of any one of items 46 to 49, wherein the electric pulse generator is configured to generate an immediate raise of amplitude. The device of any one of items 46 to 50, wherein the electric pulse generator is configured to generate one or more electric pulse(s) that is/are rectangular shape, square shaped, triangle shaped, sawtooth shaped or L-shaped. The device of any one of items 46 to 51 , wherein the electric pulse generator is configured to generate one or more electric pulse(s) that is/are rectangular shaped. The device of any one of items 46 to 52, wherein the electric pulse generator is configured to generate pulsating direct current. The device of any one of items 46 to 53, wherein the electric pulse generator is configured to generate a single electric pulse. The device of any one of items 46 to 54, wherein the electric pulse generator is configured to generate multiple electric pulses. The device of any one of items 46 to 55, wherein the electric pulse generator is configured to generate a single pulse package consisting of multiple electric pulses. The device of item 54, wherein the electric pulse generator is configured to generate multiple electric pulses consisting of single pulses that have a constant voltage. The device of item 54, wherein the electric pulse generator is configured to generate multiple pulse packages consisting of single electric pulses that have constant voltage. The device of any one of items 46 or 58, wherein the electric pulse generator is configured to generate a single pulse package consisting of multiple pulses that have constant pulse frequency. The device of any one of items 46 to 59, wherein the electric pulse generator is configured to generate a single pulse package consisting of multiple pulses that have constant pulse duration time. The device of any one of items 46 to 60, wherein the electric pulse generator is configured to generate a single pulse package consisting of multiple pulses that have a duration time of about 100 pSec to about 3000 pSec, preferably about 400 pSec to about 900 pSec, each. 62. The device of any one of items 46 to 61 , wherein the electric pulse generator is configured to generate a single pulse, a single pulse package, or multiple packages or multiple pulses with a pulse or pulse package duration time of about 100 pSec to about 3000 pSec, preferably about 400 pSec to about 900 pSec.

63. The device of any one of items 46 to 62, further comprising a holding capillary.

64. The device of item 63, wherein a cathode is connected to the holding capillary.

65. Use of an electric pulse generator or an electric pulse as defined in any one of the preceding items for facilitating penetration of a cell membrane and/or cell nucleus and/or pronucleus with an injection capillary. fc * *

[0045] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0046] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0047] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0048] When used herein “consisting of’ excludes any element, step or ingredient not specified in the claim element. When used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms “comprising”, “consisting”, “essentially of’ and “consisting of’ may be replaced with either of the other two forms. Any such replacement is envisioned by the present disclosure.

[0049] Herein, the elements of the present invention are described. Though, these elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments described throughout the specification should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments, which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutation and combination of all elements described herein should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0050] The term ‘about’ is understood to mean that there can be variation in the respective value or range that can be up to 5%, up to 10%, up to 15% or up to and including 20% of the given value. For example, if a formulation comprises about 5 mg/ml of a compound, this is understood to mean that a formulation can have between 4 and 6 mg/ml, preferably between 4.25 and 5.75 mg/ml, more preferably between 4.5 and 5.5 mg/ml and even more preferably between 4.75 and 5.25 mg/ml, with the most preferred being 5 mg/ml. As used herein, an interval which is defined as “(from) X to Y” equates with an interval which is defined as “between X and Y”. Both intervals specifically include the upper limit and also the lower limit. This means that for example an interval of “5 mg/ml to 10 mg/ml” or “between 5 mg/ml and 10 mg/ml” includes a concentration of 5, 6, 7, 8, 9, and 10 mg/ml as well as any given intermediate value.

EXAMPLES

[0051] The following Examples illustrate the invention, but are not to be construed as limiting the scope of the invention.

[0052] Example 1: CRISPR/Cas9 microinjections of Prkg1(KI) RNA into mouse oocytes

[0053] In vitro fertilisation. Six female B6D2F1 superovulated mice were sacrificed. A total of 247 oocytes were isolated and kept at 37°C, 5% CO2 in 90 pl of 0,25 mM GSH in mHTF media under mineral oil. In parallel, cauda Epididimi of C57BL6 male mouse were transferred to 120 pl of TYH MBCD media and mixed. 10 pl of sperm solution was transferred to 90 pl of fresh TYH MBCD media and kept for 1 hour at 37°C, 5% CO₂. 15 pl (10-20 pl) of the diluted sperm solution was added to the oocytes and placed in incubator at 37°C, 5% CO2 for 3 h. Cells were washed 4 times with 120 pl of mHTF media and incubated for 3 hours at 37°C, 5% CO₂.

[0054] The fertilized oocytes (195) were injected with Prkgl (KI) and incubated in KSOM media overnight at 37°C, 5% CO₂. The resulting two-cell zygotes were subsequently implanted into the oviduct of four pseudo-pregnant 0.5d CD- mice.

[0055] After the Cytoplasm injection of 195 oocytes the following results were observed: 43 oocytes were lysed, 147 oocytes reached a two-cell stage, 5 oocytes had a one-cell stage, and 0 oocytes were fragmented (also summarized in table 1). [0056] CRISPR/Cas9 microinjections of RNA’s into mouse oocytes (cytoplasm): Prkg1_crRNA1 -12,5 ng/pl (-1 pmol/pl tracrRNA -22,0 ng/pl (-1 pmol/pl)

HiFi Cas9 mRNA 20 ng/pl

HiFi Cas9 protein 40 ng/pl ( 0,25 pmol/pl)

PrkgEX2templ 0,2 pmol/pl

The final buffer had 0,6mM HEPES (pH=7,5) and 2mM KOAc.

[0057] Example 2: CRISPR/Cas9 microinjections of VEGFR3_KI RNA into mouse oocytes [0058] In vitro fertilisation. Twelve female B6D2F1 superovulated mice were sacrificed. A total of 556 oocytes were isolated and kept at 37°C, 5% CO₂ in 90 pl of 0,25 mM GSH in mHTF media under mineral oil. In parallel, cauda Epididimi of C57BL6 male mouse were transferred to 120 pl of TYH MBCD media and mixed. 10 pl of sperm solution was transferred to 90 pl of fresh TYH MBCD media and kept for 1 hour at 37°C, 5% CO₂. 15 pl (10-20 pl) of the diluted sperm solution was added to the oocytes and placed in the incubator at 37°C, 5% CO2 for 3 h. Cells were washed 4 times with 120 pl of mHTF media and incubated for 3 hours at 37°C, 5% CO₂.

[0059] The fertilized oocytes (258) were injected with VEGFR3_KI and incubated in KSOM media overnight at 37°C, 5% CO₂. The resulting two-cell zygotes were subsequently implanted into the oviduct of eight pseudo-pregnant 0.5d CD- mice.

[0060] After the Cytoplasm injection of 258 oocytes the following results were observed: 32 oocytes were lysed, 225 oocytes reached the two-cell stage, 1 oocyte had a one-cell stage, and 0 oocytes were fragmented (also summarized in table 1).

[0061] CRISPR/Cas9 microinjections of RNA’s into mouse oocytes (cytoplasm): VEGFR3_crRNA1 -12,5 ng/pl (-1 pmol/pl tracrRNA -22,0 ng/pl (-1 pmol/pl)

HIFI Cas9 mRNA 20 ng/pl

HIFI Cas9 protein 40 ng/pl (0,25 pmol/pl)

VEGFR3_mut 0,2 pmol/pl

The final buffer had 0,6mM HEPES (pH=7,5) and 2mM KOAc.

[0062] Example 3: CRISPR/Cas9 microinjections of Prkgl (KI) RNA into mouse oocytes

[0063] In vitro fertilisation. Six female CD-1 superovulated mice were sacrificed. A total of 152 oocytes were isolated and kept at 37°C, 5% CO₂ in 90 pl of 0,25 mM GSH in mHTF media under mineral oil. In parallel, cauda Epididimi of CD-1 male mouse were transferred to 120 pl of TYH MBCD media and mixed. 10 pl of sperm solution was transferred to 90 pl of fresh TYH MBCD media and kept for 1 hour at 37°C, 5% CO₂. 15 pl (10-20 pl) of the diluted sperm solution was added to the oocytes and placed in incubator at 37°C, 5% CO₂ for 3 h. Cells were washed 4 times with 120 pl of mHTF media and incubated for 3 hours at 37°C, 5% CO₂.

[0064] The fertilized oocytes (137) were injected with Prkgl (KI) and incubated in KSOM media overnight at 37°C, 5% CO2. The resulting two-cell zygotes were subsequently implanted into the oviduct of five pseudo-pregnant 0.5d CD- mice.

[0065] After the Cytoplasm injection of 137 oocytes the following results were observed: 12 oocytes were lysed, 118 oocytes reached the two-cell stage, 6 oocytes had a one-cell stage, and 1 oocyte was fragmented (also summarized in table 1 ).

[0066] CRISPR/Cas9 microinjections of RNA’s into mouse oocytes (cytoplasm): Prkg1_crRNA2 -12,5 ng/pl (-1 pmol/pl tracrRNA -22,0 ng/pl (-1 pmol/pl)

HiFi Cas9 mRNA 20 ng/pl

HiFi Cas9 protein 40 ng/pl ( 0,25 pmol/pl)

PrkgEX4templ 0,2 pmol/pl

The final buffer had 0,6mM HEPES (pH=7,5) and 2mM KOAc.

[0067] Example 4: CRISPR/Cas9 microinjections of Smarcb1_R53X RNA into mouse oocytes [0068] In vitro fertilisation. Twelve female B6D2F1 superovulated mice were sacrificed. A total of 464 oocytes were isolated and kept at 37°C, 5% CO₂ in 90 pl of 0,25 mM GSH in mHTF media under mineral oil. In parallel, cauda Epididimi of C57BL6 male mouse were transferred to 120 pl of TYH MBCD media and mixed. 10 pl of sperm solution was transferred to 90 pl of fresh TYH MBCD media and kept for 1 hour at 37°C, 5% CO₂. 15 pl (10-20 pl) of the diluted sperm solution was added to the oocytes and placed in incubator at 37°C, 5% CO₂ for 3 h. Cells were washed 4 times with 120 pl of mHTF media and incubated for 3 hours at 37°C, 5% CO₂.

[0069] The fertilized oocytes (220) were injected with Smarcb1_R53X and incubated in KSOM media overnight at 37°C, 5% CO₂. The resulting two-cell zygotes were subsequently implanted into the oviduct of eight pseudo-pregnant 0.5d CD- mice.

[0070] After the Cytoplasm injection of 220 oocytes the following results were observed: 20 oocytes were lysed, 200 oocytes reached a two-cell stage, 0 oocytes had a one-cell stage, and 0 oocytes were fragmented (also summarized in table 1).

[0071] CRISPR/Cas9 microinjections of RNA’s into mouse oocytes (cytoplasm): crRNA3 “12,5 ng/pl (-1 pmol/pl tracrRNA '22,0 ng/pl (-1 pmol/pl)

Cas9 mRNA 10 ng/pl

Cas9 protein 50 ng/pl

Smarcb1_R53X_oligo 0,2 pmol/pl

The final buffer had 0,6mM HEPES (pH=7,5) and 2mM KOAc.

[0072] Example 5 Prkgl (KI) injection

[0073] In vitro fertilisation. Six female B6D2F1 superovulated mice were sacrificed. A total of 212 oocytes were isolated and kept at 37°C, 5% CO₂ in 90 pl of 0,25 mM GSH in mHTF media under mineral oil. In parallel, cauda Epididimi of C57BL6 male mouse were transferred to 120 pl of TYH MBCD media and mixed. 10 pl of sperm solution was transferred to 90 pl of fresh TYH MBCD media and kept for 1 hour at 37°C, 5% CO₂. 15 pl (10-20 pl) of the diluted sperm solution was added to the oocytes and placed in incubator at 37°C, 5% CO₂ for 3 h. Cells were washed 4 times with 120 pl of mHTF media and incubated for 3 hours at 37°C, 5% CO₂.

[0074] The fertilized oocytes (187) were injected with Prkgl (KI) and incubated in KSOM media overnight at 37°C, 5% CO2. The resulting two-cell zygotes were subsequently implanted into the oviduct of six pseudo-pregnant 0.5d CD- mice.

[0075] After the Cytoplasm injection of 187 oocytes the following results were observed: 27 oocytes were lysed, 159 oocytes reached a two-cell stage, 1 oocyte had a one-cell stage, and 0 oocytes were fragmented (also summarized in table 1 ).

[0076] CRISPR/Cas9 microinjections of RNA’s into mouse oocytes (cytoplasm):

Prkg1_crRNA2 -12,5 ng/pl (-1 pmol/pl tracrRNA -22,0 ng/pl (-1 pmol/pl)

HiFi Cas9 mRNA 20 ng/pl

HiFi Cas9 protein 40 ng/pl ( 0,25 pmol/pl)

PrkgEX4templ 0,2 pmol/pl

The final buffer had 0,6mM HEPES (pH=7,5) and 2mM KOAc.

[0077] Table 1 : Summary of the experiments

[0078] Example 6 Comparison of a electropulse induced versus a mechanical microinjection procedure

[0079] The experimental data of Figure 7 shows the comparison of the efficiencies of electropulse induced versus mechanical microinjection procedures, based on the embryo survival rates. We have investigated embryos of the B6D2F1 mouse strain, which generally survive well after mechanical microinjection procedure (Figure 7A and B), and embryos of C57BI6/J mouse strain that poorly survived after mechanical microinjections.

[0080] As shown in Figure 7C and D the survival of C57BI6/J mice after a mechanical microinjection procedure lays usually between 40% and 80%, and reaches 100% survival by the electropulse induced microinjection procedure presented herein. In both mouse lines the method of the present invention demonstrates superior increase in survival rates after injection, and exhibited improved development rates of zygotes into two-cell stage embryos (next day after injections).

[0081] Further embodiments of the invention will become apparent from the following claims.

Claims

CLAIMS A method of injecting a substance into a cell, comprising: using a capillary that is operatively connected with an electric pulse generator and filled with a liquid to be injected; and applying a single electric pulse or multiple electric pulses to said capillary; wherein the single electric pulse or multiple electric pulses are applied when penetrating the cell with said capillary. The method of claim 1, wherein the single electric pulse or multiple electric pulses are applied concurrently with penetrating the cell. The method of claim 1 or 2, wherein the substance is injected by essentially mechanical means. The method of any one of claims 1 to 3, wherein penetration is effected by a combination of mechanical stress directed to the cell by the capillary and application of said single electric pulse or multiple electric pulses. The method of any one of claims 1 to 4, wherein the electric pulse(s) has/have a duration time from about 100 pSec to about 3000 pSec, preferably about 400 pSec to about 900 pSec. The method of any one of claims 1 to 5, wherein the electric pulse(s) has/have a frequency from about 1 Hz to about 400 kHz, preferably about 40 kHz to about 100 kHz. The method of any one of claims 1 to 6, wherein the electric pulse(s) has/have an amplitude from about 0.5 V to about 400 V, preferably about 40 V to about 100 V. The method of any one of claims 1 to 7, wherein the relation of the parameters amplitude, pulse duration and number of pulses is as follows:

E*Pw*n= x wherein:

E is the amplitude in Volts;

Pw is the duration time of a single pulse or a pulse package in seconds; n is number of single pulses or pulse packages (n>1 ), wherein x has a value of at least about 10‘⁴ V*s to about 50 V*s.

26 The method of any one of claims 1 to 8, wherein multiple electric pulses in a single pulse package are applied. The method of any one of claims 1 to 8, wherein multiple electric pulse packages are applied consisting of multiple electric pulses. The method of any one of claims 1 to 10, wherein the cell is a eukaryotic cell. The method of any one of claims 1 to 11 , wherein the cell is a human cell, a mouse cell, a rat cell, a rabbit cell, a non-human primate cell, a pig cell, a camellia cell, a cow cell, a horse cell, a goat cell, a cat cell, a dog cell or a sheep cell. The method of any one of claims 1 to 12, wherein the cell comprises an oocyte, a fertilized oocyte or an embryotic cell. A microinjection device, comprising a capillary filled with a liquid comprising a substance to be injected; wherein the capillary is operatively connected to an electric pulse generator; and wherein the electric pulse generator comprises an electronic control unit that is configured to generate one single electric pulse or multiple electric pulses to facilitate the penetration of a cell or cell membrane. The device of claim 14, wherein the electric pulse generator is configured to generate one or more electric pulse(s) that has/have a duration time from about 100 pSec to about 3000 pSec, preferably about 400 pSec to about 900 pSec. The device of claim 14 or 15, wherein the electric pulse generator is configured to generate one or more electric pulse(s) that has/have a frequency from about 1 Hz to about 400 kHz, preferably about 40 kHz to about 100 kHz. The device of any one of claims 14 to 16, wherein the electric pulse generator is configured to generate one or more electric pulse(s) that has/have a/an amplitude from about 0.5 V to about 400 V, preferably about 40 V to about 100 V. Use of an electric pulse generator or an electric pulse as defined in any one of the preceding claims for facilitating penetration of a cell membrane and/or cell nucleus and/or pronucleus with an injection capillary.