Circuit arrangement for injecting nucleic acids and other biologically active molecules into the nucleus of higher eucaryontic cells using electrical current

Abstract

The invention relates to a novel circuit arrangement for electrotransfection or electrofusion, which enables the transportation of DNA and/or other biologically active molecules to the nucleus of higher eukaryotic cells or the fusion of cells, independent of cell division and with reduced cell mortality.

1 . A method for introducing nucleic acids, peptides, proteins and/or other biologically active molecules into the cell nucleus of eukaryotic cells by means of electric current or for the treatment of cells, cell derivatives, subcellular particles and/or vesicles with electric current, said method comprising:
 supplying at least one voltage pulse to the cells, cell derivatives, subcellular particles and/or vesicles,   measuring the quantity of charge supplied by the voltage pulse in at least one selectable time interval, wherein a preset desired quantity of charge is compared with the actually supplied quantity of charge, and   terminating the voltage pulse on reaching or exceeding the desired quantity of charge.   
     
     
         2 . The method of  claim 1 , wherein the supplied quantity of charge is determined from the difference between the original charge of a corresponding storage device and the residual charge. 
     
     
         3 . The method of  claim 1 , wherein a first pulse with the capacitor voltage (U 1 ) is supplied to the cells and subsequently without interruption at least one second pulse with the capacitor voltage (U 2 ) is also supplied to the cells. 
     
     
         4 . The method of  claim 3 , wherein a first pulse having a field strength of 2-10 kV/cm, a duration of 10-100 μs and a current density of at least 2 A·cm −2  is applied to the cells, and subsequently without interruption a second pulse having a current density of 2-14 A·cm −2  and a maximum duration of 100 ms is also supplied to the cells. 
     
     
         5 . The method of  claim 1 , wherein the time interval for determining the supplied charge is specified simultaneously with the supply of the charge of a first and/or preferably a second or each further pulse. 
     
     
         6 . The method of  claim 3 , wherein the switch-on time (T 2 ) of the second pulse is specified by comparing the desired quantity of charge with the actual quantity of charge supplied by the measurement time and terminated when the desired quantity of charge is reached. 
     
     
         7 . The method of  claim 1 , wherein in order to determine the actual quantity of charge a measuring cycle of 1 msec is selected, wherein during the time (T 2 ) the capacitor voltage decreases exponentially and the pulse is terminated on reaching the specified quantity of charge (Q 2 ). 
     
     
         8 . The method of  claim 1 , wherein after at least one pre-determined time interval after triggering one first and/or second pulse the flowing current is measured and, if said current exceeds or falls below a desired value, the duration of the pulse is readjusted in order to keep the supplied quantity of charge constant. 
     
     
         9 . The method of  claim 1 , wherein after at least one pre-determined time interval after triggering one first and/or second pulse the flowing current is measured and, if said current exceeds or falls below a desired value, an error message is given. 
     
     
         10 . The method of  claim 1 , wherein after at least one pre-determined time interval after triggering one first and/or second pulse the flowing current is measured and, if said current exceeds or falls below the desired value, the desired value is readjusted. 
     
     
         11 . The method of  claim 1 , wherein pre-selected setting parameters of the pulse (U 1 , T 1 , I 2 , T 2 , K 2 ) are inputted manually or by entering a code. 
     
     
         12 . The method of  claim 3 , wherein an overcurrrent cutoff for the first and second pulse is accomplished. 
     
     
         13 . The method of  claim 3 , wherein the resistance R of the cuvette used to calculate (U 2 ) is determined by a resistance measurement before triggering a power semiconductor. 
     
     
         14 . The method of  claim 3 , wherein the resistance R of the cuvette used to calculate (U 2 ) is predetermined. 
     
     
         15 . The method of  claim 3 , wherein pre-selected setting parameters of the pulse (U 1 , T 1 , I 2 , T 2 , K 2 , R) are read in via a memory card. 
     
     
         16 . The method of  claim 1 , wherein the method is used for the transfection of quiescent or dividing eukaryotic cells. 
     
     
         17 . The method of  claim 1 , wherein the method is used for the transfection of primary cells. 
     
     
         18 . The method of  claim 1 , wherein the method is used for the transfection of human blood cells. 
     
     
         19 . The method of  claim 1 , wherein the method is used for the transfection of pluripotent precursor cells of human blood. 
     
     
         20 . The method of  claim 1 , wherein the method is used for the transfection of primary human fibroblasts, endothelial cells, muscle cells or melanocytes. 
     
     
         21 . The method of  claim 1 , wherein the method is used for the fusion of cells, cell derivatives, subcellular particles and/or vesicles. 
     
     
         22 . The method of  claim 1 , wherein the method further includes using eukaryotic cells transfected according to the method for analytical or diagnostic purposes. 
     
     
         23 . The method of  claim 1 , wherein the method further includes using eukaryotic cells transfected according to the method for the manufacture of a pharmaceutical product for ex vivo gene therapy.