RU2563804C1 - Method of delivery of nucleinic acids into eukaryotic cells - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method of delivery of nucleinic acids into eukaryotic cells which provisions the transfection by the method of calcium phosphate precipitation in presence of H1.3 histone in effective quantity is offered.

EFFECT: method improves efficiency of transfection.

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Description

The invention relates to a method for introducing recombinant nucleic acids into eukaryotic cells in vitro and can be used to obtain genetically modified cells in biology, biotechnology, medicine and veterinary medicine.

A known method for the genetic modification of eukaryotic cells by the method of calcium phosphate precipitation, in which the nucleic acid forms a complex with phosphates, which is then absorbed by the cell. Calcium-phosphate method is characterized by ease of use and low cost, however, the effectiveness of this method depends on the formation of nucleic acid complexes with phosphate, which are sensitive to the pH value of solutions [1].

There is a method of increasing the efficiency of the calcium phosphate transfection method by using 2x BBS buffer pH 6.9. The method improves the efficiency of transfection from 10 to 50% [2].

Known methods for increasing the efficiency of the calcium phosphate transfection method by physically affecting target cells, for example, by ultrasound [3]. The limited use of physical methods is due to the availability of special equipment.

Physical injection methods also include microinjection [4,5]. Microinjection is a simple, economical, effective, reproducible and non-toxic method that is suitable for the delivery of large nucleic acids. The disadvantage of using microinjections is the need to manipulate with each individual cell.

The closest in essence to the invention is a method for the delivery of recombinant nucleic acids by calcium phosphate precipitation with the addition of a biodegradable polymer poly (lactide-co-glycolide) [6]. However, the use of biodegradable polymers in genetic engineering is currently under development and requires additional research.

The purpose of the invention is the creation of an effective method for the genetic modification of eukaryotic cells that does not require special equipment.

Our proposed method can significantly increase the efficiency of the calcium-phosphate transfection method and avoid the disadvantages of analogues.

The technical result is achieved in that the transfection of eukaryotic cells with the calcium phosphate method is carried out in the presence of histone in an effective amount.

By the term “histone protein (histone)” is meant various types of histones (core histones H2A, H2 B, H3, H4 and linker histone H1), including human N-bismethionine histone H1.3

An effective amount can be determined experimentally by a specialist, for example, by determining the ratio of plDNA / histone at which the complex formed does not change the electrophoretic mobility.

A brief description of the drawings.

Fig. 1. Complexation of histones / plasmid DNA in a 150 mM NaCl solution. Long exposure when photographing a gel stained with ethidium bromide. The range of the investigated quantities of histone H1.3 (with the amount of pasmid DNA 1 μg): from 0.125 to 3.0 μg. Plasmid DNA - pEGFP-N2 (BD Biosciences Clontech, Germany). A - circular relaxed DNA, B - linear DNA, C - supercoiled DNA, D - denatured DNA. M - Gene Ruller 1Kb DNA Lader marker (Thermo Fisher Scientific Inc., USA), wells 1-15 - complexes with different ratios of plasmid DNA and histone H1.3 (w: w): well 1 - 1: 3, well 2 - 1: 2.5, well 3 - 1: 2, well 4 - 1: 1.75, well 5 - 1: 1.5, well 6 - 1: 1.25, well 7 - 1: 1, well 8 - 1: 0.875, well 9 - 1: 0.75, well 10 - 1: 0.625, well 11 - 1: 0.5, well 12 - 1: 0.375, well 13 - 1: 0.25, well 14 - 1: 0.125, hole 15 - 1: 0.

Fig. 2. Flow cytofluorimetry of HEK293T cells transfected using the calcium phosphate method in combination with histonofection. 48 hours after transfection. Cells were transfected with pEGFP-N2 plDNA. The background fluorescence level was 0.73% (A). B - transfection using the calcium phosphate method. B - transfection of cells using a combined method: histonofection, ratio of pDNA / H1.3 - 1: 0.1 (w: w), and calcium phosphate. G - transfection of cells using the combined method: histonofection, the ratio of plDNA / H1.3 - 1: 3 (w: w), and calcium phosphate.

Fig. 3. Transfection of HEK293T cells using the calcium phosphate method in combination with histonofection. Cells were transfected with pEGFP-N2 plDNA. The percentage of EGFP-positive cells was counted 2 days after transfection using flow cytometry. The background fluorescence level was 0.73%. Ca-R - transfection using the calcium phosphate method. Ca-P + plDNA / H1.3 1: 0.1 - cell transfection using the combined method: histonofection, ratio

plDNA / H1.3 - 1: 0.1 (w: w), and calcium phosphate. Ca-P + plDNA / H1.3 1: 3-transfection of cells using the combined method: histonofection, the ratio of plDNA / H1.3 - 1: 3 (w: w), and calcium phosphate. The results are presented as mean ± standard deviation (n = 3). * - samples that were statistically significantly different from Ca-P (p value <0.05).

Fig. 4. Transfection of hTGSC cell culture with plasmid DNA / histone H1.3 complexes. A, C - fluorescence microscopy, B, D - phase-contrast light microscopy. 24 hours incubation after transfection. Transfection with TransFast transfection agent (A, B) or plasmid DNA / histone H1.3 complexes (B, D) in a ratio of 1: 125 (w: w).

Fig. 5. Transfection of human fibroblasts with plasmid DNA / histone H complexes 1.3. A, C - fluorescence microscopy, B, D - phase-contrast light microscopy. 24 hours incubation after transfection. Transfection with TransFast transfection agent (A, B) or plasmid DNA / histone H1.3 complexes (B, D) in a ratio of 1: 125 (w: w).

Fig. 6. Transfection of HeLa cell culture with plasmid DNA / histone H complexes 1.3. A, C - fluorescence microscopy, B, D - phase-contrast light microscopy. 24 hours incubation after transfection. Transfection with TransFast transfection agent (A, B) or plasmid DNA / histone H1.3 complexes (B, D) in a ratio of 1: 125 (w: w).

Fig. 7. Transfection of cell culture MCF7 with plasmid DNA / histone H complexes 1.3. A, C - fluorescence microscopy, B, D - phase-contrast light microscopy. 24 hours incubation after transfection. Transfection with TransFast transfection agent (A, B) or plasmid DNA / histone H1.3 complexes (B, D) in a ratio of 1: 125 (w: w).

Fig. 8. Flow cytofluorimetry of hTGSC cells transfected with plasmid DNA. Histogram of the distribution of GFP fluorescence in the cell population. A - non-transfected control (0.19% GFP-positive cells - background fluorescence); B - plasmid DNA / histone H 1.3 complexes in a ratio of 1: 125 (w: w) (0.96% GFP-positive cells); B - TransFast transfection agent (11.57% GFP-positive cells - positive control).

Fig. 9. Flow cytofluorimetry of hTGSC cells transfected with plasmid DNA. Scatter plot of cells (forward scatter, forward scatter, FSC) versus granularity (side scatter, side scatter, SSC). A - non-transfected control; B - complexes of plasmid DNA / histone H 1.3 in a ratio of 1: 125 (w: w); B - TransFast transfection agent (positive control). The physical parameters of the cells (FSC and SSC) did not change under the action of the TransFast transfection agent and plasmid DNA / histone H 1.3 complexes compared to untransfected cells.

The method is carried out sequentially, for example, in three stages.

First step. The cultivation of cells.

All work with cell culture is carried out in a sterile laminar box in accordance with generally accepted rules of work in the laboratory of the 2nd biosafety class.

Human embryonic kidney cells HEK-293T (ATCC, CRL-11268) were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum (FBS), 2 mm L-Glutamine and 1 × a mixture of antibiotics penicillin and streptomycin (manufacturer - PanEco, Russia). Cells are incubated at plus 37 ° C in a humid atmosphere containing 5% CO ₂ - Cell reseeding is carried out at a cell monolayer density of 90% using a 0.25% trypsin-EDTA solution (manufacturer - Sigma-Aldrich, USA).

In this way, the production of HEK293A and HeLa cell cultures is completed and the second stage is started.

Second phase. Preparation of a solution of recombinant histone H1.3.

To prepare recombinant histone H1.3 (histone protein), a sample of histone H1.3 “Oncogist” is made (manufacturer - IBCh RAS, Russia), dissolved in MilliQ sterile water to a concentration of 50 μg / μl. Histone H1.3 working solution is stored at plus 4 ° C.

The third stage. Transfection of HEK293T cells by calcium phosphate precipitation.

HEK293T cells are transfected with pEGFP-N2 plDNA complexes and histone H1.3 in various combinations (plDNA / H1.3 ratios (w: w) - 1: 0, 1: 0.1, 1: 3). Plasmid vector pEGFP-N2 (manufacturer - BD Biosciences Clontech, Germany) encodes the gene for improved green fluorescent protein EGFP.

The combinations were selected on the basis of experiments on the retention of plDNA / H1.3 complexes in an agarose gel. At a ratio of 1: 2-1: 3, binding of plDNA to histone occurred, leading to a complete loss of mobility of the complexes during gel electrophoresis. Correspondingly, the ratio 1: 0.1 was chosen as obviously lower, in which the plDNA / H1.3 complex did not change the electrophoretic activity (Fig. 1).

An effective ratio in this case is the mass ratio of plDNA / H1.3. 1: (0.1-3).

HEK293T cells are cultured in a 24-well plate to a cell monolayer density of 60-70%. A transfection mixture for each ratio is prepared in a 1.5 ml tube: 1 μg of plDNA and the required amount of histone H1.3 are added to 50 μl of 0.5 M CaCl ₂ . The mixture was vortexed, precipitated and incubated for 30 minutes at room temperature. After incubation, the mixture was added dropwise to a 15 ml tube containing 50 μl of a 2 × HBS solution while stirring the contents on a vortex. The mixture was incubated for 15 minutes at room temperature and added to the cells (100 μl per well). 6-8 hours after transfection, change the culture medium to fresh.

As a control, HEK293T cells are transfected with the calcium phosphate method without the addition of histone H1.3.

Transfection efficiency is determined by expression of the egfp reporter gene (pEGFP-N2 plasmid) by flow cytometry. Due to the fact that the cell population has natural heterogeneity in terms of autofluorescence, the threshold value of fluorescence was chosen so that 99.27% of the cells were considered not fluorescent, and 0.73% of the cells, respectively, were considered false positive by fluorescence (Fig. . 2A).

It was shown that the addition of histone H1.3 in relation to 1: 0.1 (w: w) plDNA to the transfection mixture increased the transfection efficiency by 41% (58% of EGFP-positive cells) (Fig. 2B) compared to cells transfected with the calcium phosphate method without the addition of histone H1 (41% EGFP-positive cells) (Fig. 2B). The addition of histone H1.3 in proportion to 1: 3 (w: w) plDNA to the transfection mixture increased the transfection efficiency by 29% (53%) of EGFP-positive cells compared to cells transfected with the calcium phosphate method without the addition of histone H1.3 (Fig. 2G).

It is worth noting that the maximum efficiency was achieved by adding a small concentration of histone, which did not lead to complete binding of plDNA, thus, the DNA continued to bear the total negative charge (ratio of plDNA / H1.3 (w: w) 1: 0.1), necessary for complexation with phosphate. At the same time, the presence of single histone H1.3 molecules on plDNA probably increased the efficiency of nuclear transport after the penetration of complexes into the cell.

The generalized research results are shown in Figure 3.

Since, using the calcium phosphate method, a wide range of mammalian cells are efficiently transfected, including (but not limited to) CHO, 293, COS, HeLa, etc. [1], and the complexation mechanism of plDNA / H1.3 and plDNA / H1.3 with phosphates is universal , the proposed method of introducing nucleic acid using histone will also be effective for transfection of other cell cultures.

Determination of the efficiency of transfection of target cells HeLa, MCF7, hTGSC (MSCs) and human fibroblasts using plasmid DNA / histone H1.3 complexes.

The gfp gene expressing the green fluorescent GFP protein (plasmid pEGFP-N2) was used as the reporter. Analysis of GFP expression was carried out using standard methods of fluorescence microscopy and flow cytometry.

Transfection of HeLa, MCF7, hTGSC (MSC) cell cultures and human fibroblasts was performed with plasmid DNA / histone H 1.3 complexes in various combinations. As a positive control, TransFast transfection agent (Promega, USA) was used according to the recommendations of the manufacturer.

Preparation of plasmid DNA / histone H1.3 complexes for transfection.

Complexes of plasmid DNA and histone H 1.3 were prepared in 150 mM saline. For this, 5 μl of histone H1.3 solution (in different concentrations) was added to a test tube containing 1 μg of plasmid DNA (pEGFP-N2) in 95 μl of saline. The mixture was vortexed, precipitated and incubated for 30 minutes at room temperature, after which it was used to transfect cell cultures.

Transfection of HeLa, MCF7, hTGSC (MSC) cell cultures and human fibroblasts with plasmid DNA / histone H1.3 complexes.

24-well culture plates were used for transfection. A cell culture with a monolayer density of 60-70% was used.

In the wells of the culture plate, the medium was changed to fresh DMEM (500 ml per well) containing 10% FBS and a 1% mixture of penicillin and streptomycin antibiotics. 100 μl of plasmid DNA / histone H 1.3 complexes were added to the medium. Expression analysis was performed 24 hours after transfection.

Transfection of cell cultures using TransFast transfection agent.

TransFast (Promega, USA) a transfection agent is a representative of the liposomal transfection method in which plasmid DNA molecules are packed into lipid visicles [7–8]. When DNA-containing liposomes come into contact with the cell membrane, the lipid layers merge and the genetic material is introduced into the cell.

24-well culture plates were used for transfection. A cell culture with a monolayer density of 60-70% was used.

To 200 μl of culture medium without serum DMEM was added 6 μl of TransFast transfection agent, the mixture was thoroughly mixed on a vortex. 1 μg of plasmid DNA was added to the mixture, the mixture was thoroughly mixed, precipitated and incubated at room temperature for 15 minutes. Then the culture medium was removed from the cells by aspiration and the transfection mixture was applied to the cells. The plates were incubated for 1 hour in an incubator at 37 ° C in a humid atmosphere containing 5% CO ₂ , after which 1 ml of complete DMEM culture medium containing 10% FBS and a 1% mixture of penicillin and streptomycin antibiotics was added to each well. Expression analysis was performed 24 hours after transfection.

The efficiency of transfection with histone and other nuclear proteins depends on the type of protein and the conditions of transfection. For example, it was shown that histone extract from the calf thymus and from chicken red blood cells is able to bind DNA and transfect various cell cultures. However, the researchers observed only slight expression of reporter genes (Schneeweiss, Buyens et al.). In in vitro experiments, histones H1 and H2A showed low transfection efficiency of both primary and transformed cell cultures (Tomita, Higaki et al. 1992; Demirhan, Hasselmayer et al. 1998). Weng et al. Showed that histone from Pyrococcus horikoshii is not effective for delivering recombinant genetic material to cell cultures of various lines (NIH 3T3, HEK293, HL-7702, HepG2, COS7, HeLa, etc.), even in the presence of Ca2 + (Weng, Liu et al. 2004). Similar results were obtained by Lucius et al. And Balicki et al. When transfecting cells with histone H1 without co-factor and in the presence of Ca2 + (Balicki and Beutler 1997; Lucius, Haberland et al. 2001). From the described experimental data, it can be concluded that the efficiency of transfection depends on the type of histone protein, not all histones are able to efficiently mediate cell transfection. Other nuclear and histone-like proteins may also be ineffective for transfecting cells both in vitro and invivo (Bottger, Vogel et al. 1988; Sawa, Suzuki et al. 1995; Namiki, Takahashi et al. 1998; Haberland, Dalluge et al. 1999).

The results of the efficiency of transfection of target cells HeLa, MCF7, hTGSC (MSC) and human fibroblasts using complexes of plasmid DNA / histone H1.3.

To study the efficiency of transfection with histone H1.3, the gfp gene expressing the green fluorescent GFP protein (plasmid pEGFP-N2, 1 μg per well) was used as the reporter. GFP protein expression analysis was performed using standard methods of fluorescence microscopy and flow cytometry. Transfection of HeLa, MCF7, hTGSC (MSC) cell cultures and human fibroblasts was performed with plasmid DNA / histone H1.3 complexes in various ratios - 1: 2.5, 1:25, 1: 125 and 1: 250 (w: w). TransFast reagent (Promega, USA) was used as a positive control. 24 hours after incubation with plasmid DNA / histone H 1.3 complexes, the degree of green fluorescence of target cells was evaluated. Green fluorescence was observed only when using plasmid DNA / histone H 1.3 complexes for transfection at a ratio of 1: 125 (Fig. 4, Fig. 5, Fig. 6, Fig. 7,). Compared to the TransFast transfection agent, the histone H1.3 transfection efficiency of all studied cell cultures was significantly lower. Flow cytometry of transfected cell cultures confirmed the low transfection efficiency with histone H1.3: when transfected with hTGSC cell culture with histone H1: 3, the percentage of GFP-positive cells was 0.96% with a background fluorescence level of 0.19% (Fig. 8, Table 1 ) For the TransFast transfection agent (positive control), the percentage of GFP-positive cells was 11.57% (Fig. 8). During transfection using both histone H1.3 and TransFast agent, the physical parameters of the cell culture did not change (Fig. 9).

The examples given show the usefulness of the invention for genetic engineering, in particular for introducing recombinant nucleic acids into eukaryotic cells in vitro. The method of introducing recombinant nucleic acids into eukaryotic cells may find application in the production of genetically modified cells in biology, biotechnology, medicine and veterinary medicine.

Information sources

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2. Chen, C. and H. Okayama (1987). "High-efficiency transformation of mammalian cells by plasmid DNA." Mol Cell Biol 7 (8): 2745-2752.

3. Hassan, M.A., I.S. Ahmed, et al. (2012). "Enhanced gene transfection using calcium phosphate co-precipitates and low-intensity pulsed ultrasound." Eur J Pharm Sci 47 (4): 768-773.

4. Wolf, D.P., J.A. Thomson, et al. (1990). "In vitro fertilization-embryo transfer in nonhuman primates: the technique and its applications." Mol Reprod Dev 27 (3): 261-280.

5. Davis, H.L., R.G. Whalen, et al. (1993). "Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression." Hum Gene Ther 4 (2): 151-159.

6. Kofron, M.D. and C.T. Laurencin (2004). "Development of a calcium phosphate co-precipitate / poly (lactide-co-glycolide) DNA delivery system: release kinetics and cellular transfection studies." Biomaterials 25 (13): 2637-2643.

7. Balicki, D. and E. Beutler (1997). "Histone H2A significantly enhances in vitro DNA transfection." Mol Med 3 (11): 782-787.

8. Bottger, M., F. Vogel, et al. (1988). "Condensation of vector DNA by the chromosomal protein HMG1 results in efficient transfection." Biochim Biophys Acta 950 (2): 221-228.

9. Demirhan, I., O. Hasselmayer, et al. (1998). "Histone-mediated transfer and expression of the HIVtat gene in Jurkat cells." J Hum Virol 1 (7): 430-440.

10. Haberland, A., R. Dalluge, et al. (1999). "Ligand-histone H1 conjugates: increased solubility of DNA complexes, but no enhanced transfection activity." Somat Cell Mol Genet 25 (4): 237-245.

11. Lucius, H., A. Haberland, et al. (2001). "Structure of transfection-active histone Hl / DNA complexes." Mol Biol Rep 28 (3): 157-165.

12. Namiki, Y., T. Takahashi, et al. (1998). "Gene transduction for disseminated intraperitoneal tumor using cationic liposomes containing non-histone chromatin proteins: cationic liposomal gene therapy of carcinomatosa." Gene Ther 5 (2): 240-246.

13. Sawa, Y., K. Suzuki, et al. (1995). "Efficiency of in vivo gene transfection into transplanted rat heart by coronary infusion of HVJ liposome." Circulation 92 (9 Suppl): 11479-482

14. Schneeweiss, A., K. Buyens, et al. "Synergistic effects between natural histone mixtures and polyethylenimine in non-viral gene delivery in vitro." Int J Pharm 400 (1-2): 86-95.

15. Tomita, N., J. Higaki, et al. (1992). "Direct in vivo gene introduction into rat kidney." Biochem Biophys Res Commun 186 (1): 129-134.

16. Weng, L., D. Liu, et al. (2004). "An archaeal histone-like protein as an efficient DNA carrier in gene transfer." Biochim Biophys Acta 1702 (2): 209-216.

Claims (1)

A method for the delivery of nucleic acids to eukaryotic cells, which involves transfection by calcium phosphate precipitation in the presence of histone H1.3 in an effective amount.

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