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

a technology of electrical current and nucleic acids, applied in the field of circuit arrangement, can solve the problems of low cell mortality, no circuit arrangement known so far, and no known circuit arrangemen

Inactive Publication Date: 2009-01-22
LONZA COLOGNE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

Achieves high transfection efficiency and low cell mortality by delivering DNA and biologically active molecules directly into the nucleus, enabling transfection of both dividing and quiescent cells with optimized pulse parameters for various cell types.

Problems solved by technology

None of the circuit arrangements for electroporation known so far is concerned with bringing DNA electrically specifically into the nucleus of higher eukaryotic cells.
None of the circuit arrangements known so far is optimised to make it possible for DNA and / or other biologically active molecules to be effectively transported into the cell nucleus with low cell mortality.

Method used

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

Examples

Experimental program
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Effect test

example 1

Transfection of Cytotoxic T Cells from Human Blood

[0039]Freshly prepared unstimulated (non-dividing) mononuclear cells from peripheral human blood (PBMC) were transfected with a vector which codes for the heavy chain of the mouse MHC class 1 protein H-2Kk. 5×106 cells together with 5 μg of vector DNA in a buffer having a high buffer capacity (48 mM×pH−1) and high ionic strength (280 mM) were placed at room temperature in a cuvette having a 2 mm interelectrode gap and transfected by a 1000 V pulse of 100 μs duration, followed by a current flow having a current density of 5 A·cm−2 and 40 ms. Immediately afterwards, the cells were washed from the cuvette using 400 μl of culture medium, incubated for 10 minutes at 37° C. and then transferred to a culture dish with pre-heated medium. After incubating for 24 h, the cells were successively incubated with digoxigenin-coupled anti-H-2Kk-antibody and Cy5-coupled anti-digoxigenin-antibody, as well as with a PerCP-coupled anti-CD8-antibody to i...

example 2

Transfection of Human Haematopoeitic Stem Cells (CD34)

[0040]CD34-positive cells were pre-enriched from freshly prepared PBMC described as in Example 1 by magnetic cell sorting. Respectively 1×104 CD34-positive cells were then mixed with 1×106 PBMCs, placed together with 5 μg H-2Kk-expression vector DNA in a buffer having a high buffer capacity (54 mM×pH−1) and high ionic strength (260 mM) at room temperature in a cuvette having a 2 mm interelectrode gap and transfected by a 1000 V pulse of 100 μs duration, followed by a current flow having a current density of 4 A·cm−2 and 20 ms duration. Immediately afterwards, the cells were washed from the cuvette using 400 μl of culture medium, incubated for 10 minutes at 37° C. and then transferred to a culture dish with pre-heated medium. After incubating for 16 h, the cells were successively incubated with phycoerythrin-coupled anti-H-2Kk-antibody, as well as with an APC-coupled anti-CD34 antibody to identify human haematopoietic stem cells a...

example 3

Transfection of Human Neonatal Dermal Fibroblasts (NHDF-Neo)

[0041]Human neonatal dermal fibroblasts (5×105 cells) together with 5 μg H-2Kk-expression vector DNA were placed in a buffer having a high buffer capacity (67 mM×pH−1) and high ionic strength (380 mM) at room temperature in a cuvette having a 2 mm interelectrode gap and transfected by a 1000 V pulse of 100 μs duration, followed by a current flow having a current density of 6 A·cm−2 and of 33 ms duration. Immediately afterwards, the cells were washed from the cuvette using 400 μl of culture medium, incubated for 10 minutes at 37° C. and then transferred to a culture dish with pre-heated medium. After incubating for 5 h, the cells were incubated with a Cy5-coupled anti-H-2Kk-antibody and analysed using a flow cytometer (FACScalibur, Becton Dickinson). The number of dead cells was determined by staining with propidium iodide. As shown in FIG. 3, 93% of the cells express the H-2Kk antigen which corresponds to a very high transf...

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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.

Description

CROSS-REFERENCE TO PRIOR APPLICATION[0001]This is a division of U.S. patent application Ser. No. 10 / 475,840, filed Oct. 21, 2003, which is the national stage of PCT / DE02 / 01489.BACKGROUND OF THE INVENTION[0002]The invention relates to a circuit arrangement 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, consisting of at least two storage devices for quantities of electric charge, each supplied by a high-voltage power supply which each have at least one power semiconductor for transferring the quantities of charge present in the storage devices into a suspension in a cuvette and at least one monitoring device for controlling the power semiconductor.[0003]Since the place of action of eukaryotic DNA is the cell nucleus, DNA supplied from outside must enter the nuc...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q3/00C12N15/09A61N1/32C12M1/00C12M1/42C12N15/82C12N15/87C12R1/91H02M9/04H03K3/015H03K3/57
CPCA61N1/0412H03K3/57C12N15/87A61N1/327C12N13/00
Inventor MULLER-HARTMANN, HERBERTRIEMEN, GUDULAROTHMANN-COSIC, KIRSTENTHIEL, CORINNAALTROGGE, LUDGERWEIGEL, MEIKECHRISTINE, RAINERLORBACH, ELKEHELFRICH, JULIANAWESSENDORF, HEIKESIEBENKOTTEN, GREGOR
Owner LONZA COLOGNE
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