Method of detection and quantification of exogenous DNA internalized by cells

Abstract

Method of detection and quantification of exogenous DNA internalized by cells The present invention relates to a methodology for detection and quantification of exogenous DNA internalized by eukaryotic cells. In particular, it involves labeling the DNA to be transfected with a fluorochrome and quantifying the same through flow cytometry after the transfection process, resulting in a rapid prospection of the efficiency of this transfection, quantification of the percentage of transfected cells and the intensity of fluorescence in the transfection. analyzed sample.

Description

1 / 12 Method for detecting and quantifying exogenous DNA internalized by cells. Descriptive Report FIELD OF THE INVENTION

[01] The present invention relates to the quantification of exogenous DNA internalization in cells that lack gene expression machinery and, therefore, make it difficult to assess transfection efficiency. More particularly, it relates to the protocol for labeling exogenous DNA with a fluorochrome, allowing its detection and quantification within transfected cells.

[02] In particular, the present invention relates to a process involving the labeling of the DNA to be transfected, the protocol for internalization by the cell and its quantification by flow cytometry, resulting in a rapid assessment of transfection efficiency, quantification of the percentage of cells transfected, the fluorescence intensity in the analyzed sample, and finally, the use of the cell in subsequent processes, which is not possible with currently used methods. Therefore, the present methodology is an innovative process. Petition 870250113830, dated 10 / 12 / 2025, pages 40 / 53 2 / 12 FUNDAMENTALS OF THE INVENTION From DNA transfection

[03] Gene manipulation techniques allow exogenous DNA to be introduced into eukaryotic cells, in a process called transfection. In most cell lines derived from eukaryotic organisms, once integrated into the cell's genome, the genes contained in the exogenous DNA can be expressed and biologically active proteins synthesized.

[04] The transfection process enables a range of biotechnological applications, such as: large-scale production of proteins of biological interest; insertion of genes so that the cell acquires a desired characteristic; elucidation of the molecular mechanisms involved in the control of gene expression; in vivo application, such as DNA vaccines; and production of transgenic animals through the use of reproductive biotechniques.

[05] In order to improve and analyze the efficiency of the transfection process, it is necessary to employ methods capable of detecting exogenous DNA internalized by cells. Methods for detecting DNA transfection

[06] One of the most widely used methods for detecting transfected exogenous DNA is the use of exogenous DNA containing the Petition 870250113830, dated 10 / 12 / 2025, pp. 41 / 53 3 / 12 gene related to green fluorescent protein (GFP), serving as a marker for gene transfer and expression in various organisms.

[07] This protein, when excited by ultraviolet light (395-470nm), emits a green fluorescence (509nm) that is easily monitored, without the need for additional cofactors, substrates or labels. Thus, the expression of the GFP protein can be detected directly and simply using flow cytometry and fluorescence microscopy.

[08] However, germline cell lines, such as spermatozoa, do not possess cellular machinery capable of expressing genes contained in exogenous DNA and consequently producing / synthesizing heterologous protein, making it impossible to analyze and monitor the transfection process.

[09] Among the technologies currently available for analyzing a large number of cells in a short period of time, FACS (fluorescence-activated cell sorting) flow cytometry stands out for being multiparametric, using automated analyses, and performing individual cell differentiation through the prior use of fluorochromes.

[10] Thus, it provides quick and accurate information by identifying numerous intrinsic or extrinsic characteristics contained in the cells analyzed, and distinguishing Petition 870250113830, dated 10 / 12 / 2025, pages 42 / 53 4 / 12 accurately determine organelle size and complexity by reading the intensity of fluorescence reflected in cells previously labeled with or containing molecules covalently linked to fluorochromes.

[11] In this way, the fluorescence-coupled flow cytometry technique allows the monitoring and analysis of transfection efficiency in cells without cellular machinery for heterologous gene expression, once they have been transfected with DNA labeled with a fluorochrome capable of having its fluorescence detected and quantified. OBJECTIVES OF THE INVENTION

[12] There is a lack of ways to quickly and accurately quantify the efficiency of the exogenous DNA transfection process in cell lines used in the field of scientific research in the state of the art.

[13] To meet these requirements, it is necessary to develop an effective method that, as a result, detects and quantifies exogenous DNA internalized by cells, without damaging them, to be used in subsequent processes.

[14] In view of the foregoing, the present invention aims to provide a method for quantifying exogenous DNA internalized by eukaryotic cells in vitro. Petition 870250113830, dated 10 / 12 / 2025, pp. 43 / 53 5 / 12 DETAILED DESCRIPTION OF THE INVENTION

[15] The present invention relates to a method that uses a flow cytometry device to detect and quantify the percentage of cells transfected with exogenous DNA labeled with fluorochrome Cyanine-3.

[16] This quantification method can be used for different cultured and manipulated cell lines in vitro that are transfected with between 0.1 and 1 pg (100 to 1000 ng) of Cyanin-3 labeled DNA.

[17] Preferably, the method can be carried out following the following protocol: the labeling of exogenous DNA should be achieved through the polymerase chain reaction (PCR) technique using oligonucleotide primers containing the fluorochrome Cyanine-3 at the 5' end.

[18] The quantification of labeled DNA should be performed using the UV light spectrophotometry method to standardize the concentration of labeled DNA to be used in subsequent steps of the protocol. Cells should be incubated for up to 60 minutes under optimal culture conditions specific to the cell in question, in contact with exogenous DNA labeled with a fluorochrome. After the transfection period, the cells should be treated with a solution containing 1U of DNase enzyme for minutes at a temperature of 22°C for complete removal of the DNA. Petition 870250113830, dated 10 / 12 / 2025, pages 44 / 53 6 / 12 exogenous free present in the medium and bound to the outer part of the cell membrane.

[19] After treatment with DNase, the transfected cells should be washed three successive times by centrifugation at 900xg for 5 minutes to completely remove the DNase enzyme and undigested exogenous DNA. Prior to flow cytometry analysis, the cells should have their genomic DNA stained; for this, they will be incubated for 5 minutes in the presence of a fluorescent double-stranded DNA intercalating probe.

[20] After the genomic DNA labeling period, the cells should be analyzed in a flow cytometry instrument with the appropriate settings for detection of exogenous DNA labeled with fluorochrome, quantification of fluorescence intensity and quantification of the percentage of cells containing internalized exogenous DNA. EXAMPLES

[21] To allow a better understanding of the present invention and to clearly demonstrate the technical advances achieved, examples of possible combinations for this invention are now presented. According to the potential applications of the present invention and by way of illustration only, without intending to limit in any way the scope of the present invention, they are Petition 870250113830, dated 10 / 12 / 2025, pages 45 / 53 7 / 12 described below are possible uses for the method of quantifying exogenous DNA internalized by eukaryotic cells through flow cytometry. EXAMPLE 1 - Detection and quantification of exogenous DNA internalized by sexed bovine sperm cells.

[22] Aiming at the production of sex-predefined transgenic bovine embryos, sexed sperm cells transfected with exogenous DNA are used in the in vitro fertilization process. For practical purposes, only 1x106 (1 million) spermatozoa and 0.1 pg of labeled exogenous DNA will be used in this example; however, a larger quantity of cells and exogenous DNA can be employed, depending on the researcher's demand and needs.

[23] First, straws containing sperm cells should be thawed in water pre-warmed to 36.5°C for 30 seconds and washed twice by centrifugation to remove the supernatant containing cryoprotectants and undesirable somatic cells. By counting in a Neubauer chamber, the sperm concentration should be adjusted to 1x106 cells, which will be maintained in Opti-MEM I motility maintenance medium in a final volume of 200 µl (200 microliters). Petition 870250113830, dated 10 / 12 / 2025, pages 46 / 53 8 / 12

[24] The exogenous DNA to be transfected must be labeled by means of a polymerase chain reaction using oligonucleotide primers containing the covalently linked fluorochrome, with the reaction product being the exogenous DNA containing the fluorochrome in its composition. Afterwards, it must be quantified by means of UV light spectrophotometry and its concentration adjusted between 0.1 gg and 10 gg.

[25] After the preparation of the sperm cells and the labeling of the exogenous DNA, the sperm transfection process should be carried out by incubating 1x106 spermatozoa in contact with 100 ng of exogenous DNA for 60 minutes at a temperature of 36.5°C. Immediately afterwards, the transfected sperm cells should be subjected to treatment with a solution containing 1U of DNase for 15 minutes at an ambient temperature of 23°C.

[26] After the DNase treatment step to completely remove free exogenous DNA in the medium or anchored to the outer part of the sperm membrane, the cells should be washed twice by centrifuging for 5 min at 900 χ g to inactivate and remove the DNase enzyme present in the medium.

[27] Prior to analysis by flow cytometry, the cells must have their genomic DNA stained to enable total counting by flow cytometry. Petition 870250113830, dated 10 / 12 / 2025, pages 47 / 53 9 / 12 flow. For this, they will be incubated for 10 minutes in the presence of 1 μl of a stock solution at a concentration of 1.7 μM of Hoechst 33342, which is a double-stranded DNA intercalating agent, staining all the chromatin of the cell.

[28] After the genomic DNA staining period, the cells should be analyzed in a flow cytometry instrument for detection of exogenous DNA labeled with the fluorochrome in question, resulting in quantitative data regarding the quantification of the fluorescence intensity emitted in the analyzed sample (Fig. 1) and the percentage of sperm cells containing the labeled exogenous DNA internalized (Fig. 2).

[29] From this point, the cells can be recovered after analysis and used in a subsequent process, such as in vitro fertilization to generate bovine embryos containing exogenous DNA, from the transfected sperm cell, inserted into their genome. EXAMPLE 2 Detection and quantification of the internalization of exogenous DNA-liposome complexes in bovine spermatozoa.

[30] One of the most widely used methods to increase the efficiency of the transfection process is the use of cationic liposomes in association with exogenous DNA to be internalized by cells. Consequently, a greater Petition 870250113830, dated 10 / 12 / 2025, pages 48 / 53 10 / 12 the amount of internalized exogenous DNA reflects a greater success of the biotechnology applied to animal transgenesis used.

[31] For practical purposes, 2x106 (2 million) spermatozoa and 1 pg of labeled exogenous DNA will be used in this example; however, a larger quantity of cells and exogenous DNA may be employed, depending on the lipofection protocol and the researcher's needs. First, straws containing the sperm cells should be thawed in water pre-warmed to 36.5°C for 30 seconds and washed twice by centrifugation to remove the supernatant containing cryoprotectants and undesirable somatic cells.

[32] By counting in a Neubauer chamber, the sperm concentration should be adjusted to 2x106 cells, which will be maintained in Opti-MEM I motility maintenance medium in a final volume of 200 pl.

[33] Concomitantly, the exogenous DNA to be transfected must be labeled by means of a polymerase chain reaction using oligonucleotide primers containing the fluorochrome at one of its ends (5' or 3'), with the reaction product being the exogenous DNA containing the fluorochrome in its composition. After that, it must be quantified by means of UV light spectrophotometry and its concentration adjusted to 1 pg (1000 ng). Petition 870250113830, dated 10 / 12 / 2025, pp. 49 / 53 11 / 12

[34] Prior to transfection, exogenous DNA-cationic liposome complexes should be formed by incubating 1 μg of labeled exogenous DNA with 2 μl of the commercial cationic liposome stock solution Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) for 15 minutes at room temperature of 23°C.

[35] After the preparation of the sperm cells and the exogenous DNA-cationic liposome complexes, the sperm transfection process should be carried out by incubating the 2x106 sperm cells in contact with 1000 ng of exogenous DNA for 60 minutes at a temperature of 36.5°C. Immediately afterwards, the transfected sperm cells should be treated with a solution containing 1U of DNase for 15 minutes at an ambient temperature of 23°C.

[36] After the DNase treatment step to completely remove free exogenous DNA in the medium or anchored to the outer part of the sperm membrane, the cells should be washed twice by centrifuging for 5 min at 900xg to inactivate and remove the DNase enzyme present in the medium.

[37] Prior to flow cytometry analysis, the cells must have their genomic DNA stained to enable total counting by flow cytometry. For this, they will be incubated for 10 minutes in the presence of 1 μl of a stock solution in Petition 870250113830, dated 10 / 12 / 2025, pages 50 / 53 12 / 12 concentration of 1.7 μM of Hoechst 33342, which is a double-stranded DNA intercalating agent, staining all the chromatin of the cell.

[38] After the genomic DNA staining period, the cells should be analyzed in a flow cytometry instrument for detection of exogenous DNA labeled with the fluorochrome in question and quantification of fluorescence intensity (Fig. 3) and the percentage of sperm cells containing the labeled exogenous DNA-cationic liposome complex internalized (Fig. 4). Petition 870250113830, dated 10 / 12 / 2025, pages 51 / 53

Claims

1 / 1 CLAIMS 1. A method for labeling and quantifying exogenous DNA internalized in sperm cells, characterized by labeling the exogenous DNA with fluorochrome Cyanine-3 via polymerase chain reaction (PCR) using oligonucleotide primers containing the fluorochrome Cyanine-3 (Cy3), and analysis of the internalization of the exogenous DNA by flow cytometry.

2. Method according to claim 1, characterized in that the PCR reagents are buffers, nucleotides, DNA polymerase, fluorochrome Cyanin-3 (Cy3) and oligonucleotides.

3. Method, according to claim 1, characterized in that the absolute and percentage quantification of transfected cells is performed with 0.1 to 1 μg (100 to 1000 ng) of exogenous DNA labeled with the fluorochrome Cyanine-3 (Cy-3). Petition 870250113830, dated 10 / 12 / 2025, p. 25 / 53

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