RU2818348C1 - Recombinant plasmid dnas px458d10a_myc, providing myc gene knockout in human cells, method of producing human cell line with myc gene knockout and human breast cancer cell line mda-mb231_myc with suppressed myc gene expression - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to recombinant plasmid DNA PX458D10A_Myc. Plasmid provides the MYC gene knockout in the human breast cancer cell lines MDA-MB231_Myc, has a molecular weight of 5.7 MDa and is a plasmid vector PX458D10A, hydrolysed at a BpiI site, containing a replication initiation site of plasmid pBR322, fragment of 20 base pairs, complementary DNA of human MYC gene, having sequence SEQ ID NO 1-4. Present invention also relates to a method for producing a human breast cancer cell line MDA-MB231 with suppressed MYCgene expression, involving transfection of human cells with plasmid DNA PX458D10A_Myc.

EFFECT: present invention provides producing recombinant plasmids providing MYC gene knockout in MDA-MB231 human breast cancer cells, and the most acceptable and cost-effective method of producing a human cell line with reduced expression of the c-Myc transcription factor for research and development.

2 cl, 4 dwg, 2 ex

Description

The invention relates to biotechnology, genetic and protein engineering, namely, to the production of a genetic construct that provides knockout of the MYC gene in order to reduce the expression of the c-Myc transcription factor in human cells, which can be used to study the mechanisms of carcinogenesis, metastasis and regeneration.

Obtaining genetic constructs for the purpose of creating model cell lines for studying the processes of cell dedifferentiation is extremely in demand in modern biomedicine, in particular in oncology and regenerative medicine.

The MYC gene is located in the 8q24.21 locus of human chromosome 8, contains 3 exons, 4 promoters _P0 , _P1 , _P2 , _P3 , two alternative start codons (CTG and ATG); therefore, there are two isoforms of MYC mRNA and two isoforms of the protein - c-Myc with a molecular weight of 64 kDa and a minor protein with a molecular weight of 67 kDa [1].

The Myc protein is a pleiotropic transcription factor that regulates the expression of many genes that affect key processes occurring in the cell such as the cell cycle, apoptosis, response to DNA damage, hematopoiesis, differentiation, and metabolism [2, 3]. The human Myc protein contains several highly conserved regions that are functionally important and are organized similarly among the three paralogs: c-MYC, n-MYC, and l-MYC.

MYC is an oncogene associated with the emergence and development of various malignant tumors. The c-Myc protein is overexpressed in more than 70% of human malignancies, including lymphomas, leukemia, carcinomas, neuroblastomas, melanomas, breast, ovarian, prostate and liver tumors. Most malignancies that demonstrate high c-Myc expression are highly aggressive and/or difficult to treat [3, 4].

The MYC gene represents one of the most promising targets for cancer treatment. It has been demonstrated that transient inhibition of MYC in vivo leads to sustained tumor regression, promoting arrest of proliferation, apoptosis and cellular senescence in cancer cells. However, the important functional domains of c-Myc are inherently disordered and lack a site of enzymatic activity, which is a barrier to drug design that could target the protein to downregulate MYC expression and prevent tumor development [3]. One way to solve this problem is to search for inhibitors that affect not the Myc protein directly, but its heterodimerization with the Max protein, an obligatory partner of Myc, without which binding of the Myc-Max dimer to gene promoters is impossible. Examples of such inhibitors are the following chemical compounds: SaJM589 [4], MYCMI-6 [5], KSI-3716 [6].

Another approach to inhibiting the action of the Myc protein is to look for inhibitors that compete with the Myc protein for binding to the promoters of its target genes, preventing their transcription, for example, Omomyc is a small protein consisting of 90 amino acids that has undergone clinical phase studies in cancer patients lung, colon and mammary gland [3].

Alternatively, approaches to reduce c-Myc expression using shRNA to the gene are proposedPOU5F1,encoding the Oct4 protein and being an enhancerMYC;or as a result gene knockoutMAPKAPK2, encoding the Mk2 kinase involved in the phosphorylation and subsequent activation of Oct4 [7].

To conduct research aimed at developing approaches to reduce c-Myc expression, the creation of model cell lines that differ in the level of expression of the MYC gene is required. Such cell lines are in high demand both for in vitro studies and for in vivo studies on human tumor xenograft models. For example, ATCC offers a research panel designed for basic research and development of anticancer drugs, consisting of 9 types of cell cultures that contain different MYC mutations or different numbers of copies of the MYC gene - Genetic Alteration Cell Panel (ATCC). The panel includes B lymphoma cell lines CRL-1647 ST486, CRL-1648 CA46 and HTB-62 P3HR-1, gastric cancer CRL-5974 SNU-16, lung cancer HTB-175 NCI-H82, CRL-2081 MSTO-211H and HTB-171 NCI-H446, leukemia CCL-240 HL-60 and plasmacytoma CRL-9068 NCI-H929.

The CRISPR/Cas genome editing system can be used to knock out the MYC gene. The CRISPR/Cas system was first described as an adaptive immune system in bacteria and archaea, and is now also a biotechnological tool for genome editing of living cells [8, 9]. One variant of CRISPR/Cas systems is the CRISPR/Cas9 system, which functions through RNA (gRNA), which forms a gRNA-Cas9 complex and directs the Cas9 nuclease, isolated, for example, from Streptococcus pyogenes (SpyCas9), to specified regions of the genome. The gRNA-Cas9 complex recognizes the target sequence, binds to it through complementary interactions, and catalyzes the cutting of double-stranded DNA.

The break activates repair through nonhomologous end joining (NHEJ) or homologous repair (HDR). In the vast majority of cases, NHEJ is activated, resulting in insertions and/or deletions that disrupt the sequence of the target gene, knocking it out. Targeting of gRNA-Cas9 to DNA is accomplished by the presence of a sequence in the gRNA that is complementary to the target gene sequence. The target sequence in the genome is usually about 20 bp in length. and must be adjacent to the PAM sequence 5'-NGG-3', which is necessary for nuclease recognition of the target site.

The technical objective of the invention is to create recombinant plasmids that provide knockout of the MYC gene in human cells and the most acceptable and cost-effective method for obtaining a human cell line with reduced expression of the c-Myc transcription factor for research and development.

The Lenti-sh1368 knockdown c-myc lentiviral vector is known, directing the expression of interfering shRNA to the c-Myc gene to inhibit c-Myc expression in mammalian cells [10]. The plasmid pX459-puro-hMYC is known for expression in mammalian cells of a guide RNA complementary to the sequence 5'-TGCTGCCAAGAGGGTCAAGT-3' of the human MYC gene [11]. The closest to the claimed technical solution in terms of technical essence and achieved technical result is the lentiviral plasmid vector pXPR_BRD003-sgMYC, which provides expression of guide RNA to the MYC gene [12]; In the original work, the vector was used as a positive control for the performance of the WRN helicase gene knockout method. This lentiviral plasmid vector provided expression of guide RNA to the sequence 5'-GACAACGTCTTGGAGCGCCAG-3' located in the coding region of the MYC gene (exon 2); knockout of the MYC gene was achieved by introducing a double-stranded cut into the DNA of the MYC gene by Cas9 nuclease, which forms a complex with this guide RNA.

It is known that the use of the CRISPR/Cas9 system is associated with the risk of introducing off-target breaks into genomic DNA. To increase the specificity of editing, a mutant form of Cas9 nuclease, Cas9D10A, or nickase, which makes a cut in only one of the double-stranded DNA strands, can be used [13]. To knock out a gene using Cas9D10A, a pair of guide RNAs are selected that are complementary to the upper and lower strands of DNA, ensuring that nickase is directed to regions of a given gene (the regions must be limited by the PAM sequence 5'-NGG-3') so that, as a result of hydrolysis by nickase, Each DNA strand formed one cut located at a distance of 40-70 bp. from each other. Vectors that allow knockout of the MYC gene using this method have not been described in the literature.

A new technical result is achieved by obtaining recombinant plasmid DNA PX458D10A_Myc, which ensures knockout of the MYC gene in human cells due to the ability to express RNA containing a 20-nucleotide long region complementary to the DNA of the second exon of the human MYC gene, and capable of forming a complex with Cas9D10A, making a cut in the DNA molecule for providing gene knockout, which are plasmid vectors PX458D10A hydrolyzed at the BbsI (BpiI) site, containing the replication initiation site of the plasmid pBR322, the gene encoding the Cas9D10A nuclease fused with the nuclear localization signal of the SV40 virus (NLS), and transcribed in mammalian cells as a single transcript, separated by the T2A sequence of the 2A peptide of the capsid of the Thosea asigna virus, with the sequence encoding the green fluorescent protein EGFP, the U6 promoter of human RNA polymerase III, a fragment of 20 bp in size, complementary to the coding sequence of the human MYC gene, expressed as part of a guide RNA molecule from Streptococcus pyogenes the CRISPR/Cas9 system and forming a complex with Cas9D10A, capable of making a cut in one of the DNA strands, the genetic marker bla β-lactamase gene, which determines resistance to ampicillin during transformation of Escherichia coli cells with a plasmid.

Also, a new technical result is a method for producing the monoclonal cell line PX458D10A_Myc, which involves transforming E. coli Stable3 cells with a pair of constructed plasmids PX458D10A_Myc, growing transformed cells in a medium with ampicillin, isolating recombinant plasmid DNA, transfecting human cells with plasmid DNA and obtaining a clone of a single cell with decreased expression of the c-Myc transcription factor.

The essence of the invention is that a pair of plasmids PX458D10A_Myc is constructed by inserting into the plasmid vector PX458D10A a DNA fragment encoding RNA complementary to the lower or upper DNA strand of the second exon of the MYC gene using the Golden Gate method, transforming Escherichia coli Stable3 cells with a ligase mixture, ensuring amplification of the corresponding recombinant plasmid, recombinant plasmid DNA is isolated; human cells are transfected with a pair of plasmid DNAs encoding guide RNAs to the upper and lower DNA strands, and a single cell clone with reduced expression of the c-Myc transcription factor is obtained.

The method is carried out as follows:

using genetic engineering methods, pairs of plasmids PX458D10A_Myc are obtained containing a DNA fragment complementary to the sense (top) or template (bottom) DNA strands of the coding region of the MYC gene and obtained by annealing two synthetic oligonucleotide sequences.

E. coli Stable3 cells were transformed with the constructed plasmids PX458D10A_Myc_t and PX458D10A_Myc_b and grown overnight in medium supplemented with ampicillin. The cells are then used to isolate plasmid DNA. Next, pairs of plasmid DNA containing participants encoding RNA complementary to the upper and lower DNA strands - PX458D10A_Myc_top and PX458D10A_Myc_bottom - are transfected into human cells. After transfection, the cells are grown in a complete nutrient medium for 24 hours, removed from the surface of the plastic, diluted to a concentration of 10 cells in 1 ml, and 100 μl are transferred into the wells of a culture plate. The next day, using microscopy, mark those wells of the plate that contain 1 cell, and culture the plates until 80% confluence is achieved. Next, the monoclonal cells are sequentially transferred into larger culture containers.

The initial genetic material for the construction of recombinant plasmids PX458D10A_Myc are:

a) plasmid vector PX458D10A, which ensures the insertion of a DNA fragment complementary to the coding region of the target gene and its expression as part of an RNA molecule that forms a complex with Cas9D10A nickase, capable of making a cut in one of the DNA strands, and the Cas9D10A gene;

b) a DNA fragment complementary to the coding region of the human MYC gene, which is obtained by annealing synthetic oligonucleotides.

The resulting plasmids of the PX458D10A_Myc series are used in pairs (Fig. 1) and are characterized by the following characteristics:

- have a molecular weight of 5.7 MDa and a size of 9292 bp;

- encodes an RNA molecule containing a fragment complementary to the upper or lower DNA strand of the coding region of the MYC gene, ensuring the assembly of the complex with Cas9D10A nickase and its positioning on exon 2 of the human MYC gene;

- consists of the following elements:

a) a DNA fragment of 20 bp in size, complementary to the upper or lower strand of the coding region of the MYC gene, located in the human genome on chromosome 8;

b) plasmid PX458D10A, which provides expression of RNA and Cas9D10A nickase in mammalian cells, which form a complex that makes a cut in one of the DNA strands of the exon 2 sequence of the human MYC gene;

- contains:

a) replication initiation site of plasmid pBR322;

b) genetic marker bla β-lactamase gene, which determines resistance to ampicillin during transformation of Escherichia coli cells;

c) a gene encoding a guide RNA molecule from Streptococcus pyogenes of the CRISPR/Cas9 system under the U6 promoter of human polymerase III;

d) a gene encoding the Cas9D10A nuclease from Streptococcus pyogenes of the CRISPR/Cas9 system, fused with a sequence encoding the nuclear localization signal of the SV40 virus, under the promoter of the chicken beta-actin gene;

Thus, two pairs of plasmid DNA PX458D10A_Myc were obtained, directing the production of Cas9D10A nickase in human cells and pairs of RNA, forming a complex with Cas9D10A nickase and ensuring double-stranded DNA cutting in exon 2 of the human MYC gene, a method of production was described and a monoclonal line of human breast cancer cells was obtained. MDA-MB231_Myc with reduced expression of the c-Myc transcription factor.

The invention is illustrated by the following drawings:

fig. 1. General diagram of the structural organization of the PX458D10A_Myc plasmid containing a DNA fragment of 20 bp in size, complementary to the coding region of the MYC gene, expressed in RNA, which forms a complex with Cas9D10A nickase and ensures binding to a DNA region in exon 2 of the human MYC gene, human polymerase III U6 promoter, replication initiation site of plasmid pBR322, genetic marker bla β-lactamase gene that determines resistance to ampicillin during transformation of Escherichia coli , gene encoding a mutant form of nuclease Cas9D10A from Streptococcus pyogenes of the CRISPR/Cas9 system, fused with a sequence encoding a signal nuclear localization of the SV40 virus, under the promoter of the chicken beta-actin gene;

fig. 2. Restriction analysis of recombinant plasmids PX458D10A_Myc using BbsI endonuclease. K - plasmid PX458D10A, M12 - molecular weight marker M12 (SibEnzym, Russia) (10000, 8000, 6000, 5000, 4000, 3000, 2500, 2000, 1500, 1000, 750, 500 bp), code of plasmids isolated from the clones selected for skinning, indicated at the top. Note. * samples of plasmids after treatment with endonuclease are marked, plasmids not treated with endonuclease are not marked;

fig. 3. Fragments of chromatograms obtained after Sanger sequencing of plasmid DNA isolated from recombinant clones (A) PX458D10A_MycGt, (B) PX458D10A_MycGb, (C) PX458D10A_MycPt, (D) PX458D10A_MycPb;

fig. 4. Analysis of the expression level of the transcription factor c-Myc in (A) cells of the original MDA-MB231 line and (B) cells of the monoclonal line MDA-MB231_Myc. Cells are stained with Anti-c-Myc antibodies (green signal), nuclei are stained with DAPI (blue signal).

Example 1. Preparation of recombinant plasmid PX458D10A_Myc

To obtain a fragment complementary to the region of exon 2 of the human MYC gene, 4 pairs of 25 nt long oligonucleotides are used.

MYC_G1_t_f 5’-CACCGGTATTTCTACTGCGACGAGG-3’

MYC_G1_t_r 5’-AAACCCTCGTCGCAGTAGAAATACC-3’

MYC_G2_b_f 5’-CACCGGTCGAGGTCATAGTTCCTGT-3’

MYC_G2_b_r 5’-AAACACAGGAACTATGACCTCGACC-3’

MYC_P1 _t_f 5’-CACCGCTATGACCTCGACTACGACT-3’

MYC_P1 _t_r 5’-AAACAGTCGTAGTCGAGGTCATAGC-3’

MYC_P2_b_f 5’-CACCGTTGGTGAAGCTAACGTTGAG-3’

MYC_P2_b_r 5’-AAACCTCAACGTTAGCTTCACCAAC-3’

Prepare a reaction mixture containing 10 μM of each primer from the pair, T4 ligase buffer (Evrogen, Russia), 10 u.a. T4 polynucleotide kinase (NEB, UK). The reaction is carried out in a thermal cycler (Applied Biosystems, USA) at 37°C for 30 minutes, then at 95°C for 5 minutes and the mixture is cooled at a rate of 5°C/min to 25°C. The reaction mixture is diluted ten times with water, 1 μl is taken and mixed in Quick ligation buffer (Evrogen) with the addition of 0.25 mg/ml bovine serum albumin (Sigma), 10 u.a. restriction endonucleases BpiI (BbsI), (Thermo Scientific), 375 u.a. T7 ligase (NEB), 25 ng plasmid DNA PX458D10A. The mixture is incubated in a thermal cycler (Applied Biosystems, USA) for 15 cycles, including 2 steps: 37°C for 5 min, 20°C for 5 min. The resulting reaction mixture is used to transform competent Escherichia coli Stbl3 cells. Transformed cells are cultured on Petri dishes on LB agar medium with the addition of ampicillin to a final concentration of 50 μg/ml overnight at 30°C. To isolate recombinant plasmids, 3 colonies grown on a selective medium are selected, transferred to 5 ml of LB medium (with the addition of ampicillin 50 μg/ml) and incubated overnight at 30°C with constant stirring at 180 rpm. Isolation of plasmid DNA is carried out using the Plasmid Miniprep kit (Evrogen, Russia) according to the manufacturer’s instructions.

Restriction analysis of isolated plasmids is performed using restriction endonuclease BsuI (SibEnzyme, Russia), the reaction is carried out for 1 hour at 37°C. DNA fragments obtained after endonuclease digestion are separated by electrophoresis in a 1% agarose gel with the addition of the intercalating dye ethidium bromide. Plasmid DNA samples after restriction are visualized using the G-box gel documentation system (Syngen, UK). As a result of successful cloning of the fragment encoding the guide RNA into the PX458D10A plasmid, the recognition sites for the BbsI restriction endonuclease will be removed and bands corresponding to the linear, circular and supercoiled forms of the plasmid will be detected on the electropherogram (Fig. 2).

The correspondence of the sequence of the fragment cloned into the plasmid vector PX458D10A to the expected one is confirmed by Sanger sequencing using the primer CRISPR-Seq_F 5'-GAGGGCCTATTTCCCATGATTCCTTCATAT-3' (Fig. 3).

Example 2. Generation of a human monoclonal cell line with a knockout of the MYC gene

Human breast adenocacinoma cells MDA-MB231 (ECACC) are cultured in complete nutrient medium DMEM/F12 (Gibco, UK) supplemented with 10% FBS (Gibco, UK) and 1/100 GlutaMAX 100× (Gibco, UK) in CO ₂ - incubator MCO-5M (Sanyo, Japan) at 5% CO ₂ , 37°C. Before starting an in vitro study, cells are cultured for at least 3 passages. To remove cells from plastic during subculture, TrypLE (Thermo Fisher Scientific, Denmark) is used. The number of living cells is determined by staining with Trypan Blue stain 0.4% (Invitrogen, USA) on a Countess II FL automatic counter (Thermo Fisher Scientific, Singapore).

MDA-MB231 cells are seeded into wells of a 6-well plate. The next day, the medium is removed and 2 ml of complete nutrient medium DMEM/F12 is added. Prepare a mixture for transfection: for this, 2 μl of Metafectene (Biontex, Germany) is transferred into 45 μl of 1× phosphate-buffered saline (PBS). Add 50 µl of PBS containing 2 µg of plasmids PX458D10A_Myc_Gt and PX458D10A_Myc_Gb (or PX458D10A_Myc_Pt and PX458D10A_Myc_Pb) in a mass ratio of 1: 1. The mixture is gently mixed by pipetting, incubated for 15 min at room temperature and dropwise m are added to the corresponding wells of the plate with MDA-MB231 cells.

24 hours after transfection, the cells are removed from the surface of the plastic, and a cell suspension is prepared in complete nutrient medium DMEM/F12 by serial dilution to a concentration of 10 cells/ml and dispense 100 µl into the wells of a 96-well plate. The plates are incubated in CO₂ incubator at 37°C. 24 hours after cell seeding, wells in which only 1 EGFP-expressing cell is detected when analyzed using a Leica DMi8 inverted fluorescence microscope (Leica Microsystems GmbH, Germany) are noted. Cells are cultured under standard conditions; the medium is replaced as necessary, but at least twice a week. After reaching 70-80% confluency, cells of the monoclonal line MDA-MB231_Myc in the marked wells are sequentially transplanted into 24- and 6-well plates, and then into a T25 culture flask.

Expression of the MYC gene in cells of the monoclonal line MDA-MB231_Myc is assessed by immunofluorescence analysis using Anti-c-Myc antibodies (1:100, Recombinant Anti-c-Myc rabbit monoclonal [Y69] antibody ab32072, Abcam, UK) for staining (Fig. 4). The absence of antibody staining of cells indicates a knockout of the MYC gene in them.

List of sources used

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2. Ahmadi, S.E., et al., MYC: a multipurpose oncogene with prognostic and therapeutic implications in blood malignancies. J Hematol Oncol., 2021. 14: p.121.

3. Llombart, V. and M.R. Mansoura, eBioMedicine, 2022. 75: p.103756.

4. Choi, S.H., et al., Targeted Disruption of Myc-Max Oncoprotein Complex by a Small Molecule. ACS Chem Biol., 2017. 12: p.2715-2719.

5. Castell, A., et al., A selective high affinity MYC-binding compound inhibits MYC:MAX interaction and MYC-dependent tumor cell proliferation. Sci Rep., 2018. 8: p.10064.

6. Jeong, K.C., et al., Intravesical instillation of c-MYC inhibitor KSI-3716 suppresses orthotopic bladder tumor growth. J Urol., 2014 191: p.510-518.

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Claims (2)

1. Recombinant plasmid DNA PX458D10A_Myc, providing knockout of the MYC gene in human breast cancer cell lines MDA-MB231_Myc, having a molecular weight of 5.7 MDa and representing a plasmid vector PX458D10A, hydrolyzed at the BpiI site, containing the origin of replication of the plasmid pBR322, a fragment of the size 20 bp, complementary to the DNA of the human MYC gene, having the sequence SEQ ID NO 1-4, which is expressed under the control of the U6 promoter of human polymerase III as part of a guide RNA from Streptococcus pyogenes of the CRISPR/Cas9 system, capable of forming secondary structures that ensure RNA interaction with Cas9 nuclease, as well as with its mutant form - Cas9D10A nickase, the gene encoding Cas9D10A nickase, genetic marker: bla β-lactamase gene, which determines resistance to ampicillin. 2. Method for obtaining a human breast cancer cell line - line MDA-MB231 with suppressed gene expressionMYC, which consists in carrying out transfection of human cells with plasmid DNA PX458D10A_Myc according to claim 1, a clone of a single cell with suppressed gene expression is obtainedMYC by the limiting dilution method.