Bovine TXNIP gene double-target knockout vector as well as construction method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a cattle TXNIP gene double-target knockout vector as well as a construction method and application thereof. The TXNIP gene double-target knockout vector comprises a lentiviral expression vector, a first sgRNA sequence and a second sgRNA sequence, the first sgRNA sequence is connected to the lentiviral expression vector and targets a bovine TXNIP coding region, the second sgRNA sequence is connected to the lentiviral expression vector and targets the TXNIP coding region, and nucleotide sequences of an upstream primer and a downstream primer corresponding to the first sgRNA sequence are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively. The nucleotide sequences of the upstream primer and the downstream primer corresponding to the second sgRNA sequence are respectively as shown in SEQ ID NO.3 and SEQ ID NO.4. Compared with a single-target knockout vector, the cattle TXNIP gene double-target knockout vector has higher flexibility, the targeting efficiency can be improved, and the off-target effect is reduced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a bovine TXNIP gene double-target knockout vector, a construction method and an application thereof. Background Art

[0002] Thioredoxin interacting protein (TXNIP) is a type of thioredoxin binding protein. It plays an important role in mediating oxidative stress, inhibiting cell proliferation, inducing cell apoptosis and other physiological activities by inhibiting the function of the thioredoxin system. TXNIP is a core molecule in the inflammatory process of insulin. This inflammation can lead to the death of insulin-producing cells in the human pancreas. TXNIP participates in the occurrence of type 2 diabetes by regulating NLRP3. Studies have shown that the expression of TXNIP is reduced in a variety of cancer cells, and is closely related to melanoma, hepatocellular carcinoma, breast cancer, leukemia, etc. It is a tumor suppressor-related gene. TXNIP participates in the body's glucose metabolism, lipid metabolism, immune and inflammatory responses, thereby regulating cell death, growth and differentiation.

[0003] In the related technology, Zhang Huanhuan et al. used TALEN technology to prepare a TXNIP gene knockout mouse model. Specifically, they designed a TALEN recognition splicing site on the first exon of TXNIP, constructed a TALEN vector and verified its splicing activity at the cellular level, transcribed TALENs into mRNA in vitro and injected it into the fertilized eggs of C57BL / 6J mice through fiber injection technology, and performed DNA-level identification on the F0 generation of mice to obtain TXNIP knockout mice.

[0004] However, the construction of a knockout model using the above method inevitably requires the tedious operation of synthesizing and assembling a protein module with specific DNA recognition capabilities. The construction process of the DNA recognition module of TALEN technology is labor-intensive and has high experimental costs; the knockout efficiency is not high; and the cytotoxicity is high. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a bovine TXNIP gene dual-target knockout vector, a construction method and an application thereof.

[0006] To achieve the above object, the technical solution of the present invention is:

[0007] In the first aspect, the present invention provides a bovine TXNIP gene dual-target knockout vector, comprising a lentiviral expression vector, a first sgRNA sequence targeting the bovine TXNIP coding region connected to the lentiviral expression vector, and a second sgRNA sequence targeting the TXNIP coding region connected to the lentiviral expression vector, wherein the nucleotide sequences of the upstream primer and the downstream primer corresponding to the first sgRNA sequence are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively, and the nucleotide sequences of the upstream primer and the downstream primer corresponding to the second sgRNA sequence are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0008] Optionally, the first sgRNA sequence targets the sgRNA56 site in the bovine TXNIP coding region.

[0009] Optionally, the second sgRNA sequence targets the sgRNA231 site in the bovine TXNIP coding region.

[0010] Optionally, the lentiviral expression vector is selected from the lentiviral expression vector PLV.

[0011] Optionally, the lentiviral expression vector PLV is selected from the lentiviral expression vector pLV3-U6-MCS-sgRNA-Cas9-EGFP-Puro (pLV3).

[0012] In a second aspect, the present invention provides a method for constructing the bovine TXNIP gene dual-target knockout vector as described above, comprising the following steps:

[0013] S1. Annealing the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2 to obtain a first annealing product;

[0014] S2. Annealing the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain a second annealing product;

[0015] S3. connecting the first annealing product into a lentiviral expression vector linearized after being digested with restriction endonuclease Esp3I to obtain a first single target knockout vector;

[0016] Amplifying the first single target knockout vector and the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6 to obtain an amplified product;

[0017] S4. connecting the second annealing product into a lentiviral expression vector linearized after being digested with restriction endonuclease Esp3I to obtain a second single target knockout vector;

[0018] Digesting the second single target knockout vector with restriction endonuclease EcoR I to obtain a digestion product;

[0019] S5. Connect the amplified product and the enzyme-digested product to obtain the bovine TXNIP gene double-target knockout vector.

[0020] Optionally, in step S1, the annealing includes: treating at 95°C for 2 minutes, then decreasing the temperature by 0.1°C every 8 seconds to 25°C for about 90 minutes, and temporarily storing at 4°C.

[0021] Optionally, in step S1, the molar ratio of the nucleotide sequence shown in SEQ ID NO.1 to the nucleotide sequence shown in SEQ ID NO.2 is 1:1.

[0022] Optionally, in step S2, the annealing includes: treating at 95°C for 2 minutes, then decreasing the temperature by 0.1°C every 8 seconds to 25°C for about 90 minutes, and temporarily storing at 4°C.

[0023] Optionally, in step S2, the molar ratio of the nucleotide sequence shown in SEQ ID NO.3 to the nucleotide sequence shown in SEQ ID NO.4 is 1:1.

[0024] Optionally, in step S3, the molar ratio of the first annealing product to the linearized lentiviral expression vector is 3-10:1.

[0025] Optionally, in step S5, the mass ratio of the amplification product to the enzyme cleavage product is 50-100:50-100.

[0026] In a third aspect, the present invention provides a cell line, which is obtained by co-transfecting 293FT cells with the bovine TXNIP gene dual-target knockout vector as described above and / or the bovine TXNIP gene dual-target knockout vector constructed according to the method as described above and pMD2.G and pSPAX2 plasmids for lentivirus packaging, collecting the viral supernatant 48 hours later and infecting bovine mammary epithelial cells, and selecting with puromycin.

[0027] In a fourth aspect, the present invention provides the use of the bovine TXNIP gene dual-target knockout vector as described above and / or the bovine TXNIP gene dual-target knockout vector constructed according to the method as described above and / or the cell line as described above in the study of bovine TXNIP function.

[0028] The beneficial effects of the present invention are:

[0029] Compared with the single-target knockout vector, the dual-target knockout vector of the present invention has higher flexibility, can improve the targeting efficiency and reduce the off-target effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the sequencing result diagram of the TXNIP gene single target knockout vector (pLV3-TXNIP-KO-sgRNA56 sequence);

[0031] Figure 2 This is the sequencing result diagram of the TXNIP gene single target knockout vector (pLV3-TXNIP-KO-sgRNA231 sequence);

[0032] Figure 3 This is the sequencing result of the bovine TXNIP gene double-target knockout vector;

[0033] Figure 4 This is a graph showing the protein expression level detection results of the bovine TXNIP gene. DETAILED DESCRIPTION

[0034] The examples are provided to better illustrate the content of the present invention, but the content of the present invention is not limited to the examples. Therefore, those skilled in the art can make non-essential improvements and adjustments to the implementation scheme according to the above content of the invention, which still fall within the protection scope of the present invention.

[0035] (I) Construction of TXNIP gene single target knockout vector

[0036] (1) Synthesis and annealing of sgRNA

[0037] The sequence of the CDS region (i.e., coding region) of the bovine TXNIP gene was obtained from the NCBI website. Using the CRISPOR website, two pairs of sgRNA sequences as shown in Table 1 were designed by comprehensively considering information such as specificity, cutting efficiency, and potential off-target sites. Shanghai Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the corresponding sequences. After synthesis, sgRNA annealing was performed according to the instructions of Annealing Buffer for DNA Oligos (5×). Specifically, the nucleotide sequence shown in SEQ ID No.1 and the nucleotide sequence shown in SEQ ID No.2 were used as upstream primers and downstream primers, respectively, and annealed according to the system shown in Table 2; the nucleotide sequence shown in SEQ ID No.3 and the nucleotide sequence shown in SEQ ID No.4 were used as upstream primers and downstream primers, respectively, and annealed according to the system shown in Table 2; the annealing conditions of the two reaction systems were: first treated at 95°C for 2min, then decreased by 0.1°C every 8s to 25°C for about 90min, and temporarily stored at 4°C to synthesize sgRNA56 and sgRNA231.

[0038] Table 1 sgRNA sequence list

[0039]

[0040]

[0041] Table 2 Annealing system

[0042] Reagents Dosage AnnealingBufferforDNAOligos(5×) 4μL Upstream primer (concentration 50 μM, prepared with ultrapure water) 4μL Downstream primer (concentration of 50 μM, prepared with ultrapure water) 4μL <![CDATA[ddH2O]]> 8μL

[0043] (2) Vector linearization

[0044] The pLV3 vector plasmid was digested with restriction endonuclease Esp3I. The reaction system was shown in Table 3. The reaction conditions were: incubation at 37°C for 5 min, followed by treatment at 65°C for 10 min to inactivate the enzyme, to obtain a linearized pLV3 vector.

[0045] Table 3 Vector restriction enzyme digestion system

[0046] Reagents Dosage pLV3 plasmid 1 μg 10×FastDigest 2μL Dithiothreitol DTT (20mM) 1μL Esp3I 1μL <![CDATA[ddH2O]]> Add to 20 μL

[0047] (3) Connection

[0048] The linearized pLV3 vector was connected with the sgRNA annealing product sgRNA56 and the sgRNA annealing product sgRNA231 respectively using T4 DNA ligase. The connection system is shown in Table 4. The reaction conditions of the two connection systems are: overnight treatment at 16°C for 16 hours to obtain the connection product (i.e., recombinant plasmid).

[0049] Table 4 Connection system

[0050]

[0051] (4) Conversion

[0052] The obtained ligation products were transferred into competent cells respectively. The specific steps are as follows:

[0053] ① Place 100 μL TOP10 competent cells in a 1.5 mL EP tube, add 10 μL ligation product, pipette up and down to mix, and place in an ice bath for 3 minutes;

[0054] ② Place the EP tube in a water bath at 42°C for 90 seconds, then quickly transfer to ice and cool for 2-3 minutes;

[0055] ③ Add 500 μL of LB medium without ampicillin to the EP tube, mix well, and culture on a shaker at 37°C and 200 rpm / min for 45 min;

[0056] ④Pipette 40 μL of transformed TOP10 competent cells and spread them on LB solid medium containing 100 ng / mL ampicillin;

[0057] ⑤Use a spreader to spread the cells evenly and place the plate in a 37°C incubator for 16 hours;

[0058] ⑥ Pick a single colony and culture it in 30mL of liquid culture medium containing 100ng / mL ampicillin at 37℃ and 200rpm / min for 16h, then centrifuge it at 4500rpm for 10min, discard most of the culture medium, resuspend the bacterial pellet, preserve the bacteria, and send the bacterial solution to Shanghai Shenggong Biological Company for sequencing. The sequencing results are as follows Figure 1 and Figure 2 shown.

[0059] The obtained recombinant plasmid was cloned positive after identification. This result shows that the present invention successfully constructed a knockout TXNIP gene sgRNA expression plasmid.

[0060] (II) Construction of the bovine TXNIP gene dual-target knockout vector

[0061] (1) Extraction of single target knockout plasmid

[0062] The operation steps refer to the "Omega Endotoxin-free Plasmid Extraction Instructions", the specific steps are as follows:

[0063] 1. Inoculate the above positively identified recombinant plasmid bacterial suspensions into 50 mL of LB liquid culture medium containing 100 ng / mL ampicillin, and culture at 37°C in a shaking incubator for 16 h;

[0064] 2. Take 10-15 mL of bacterial solution and centrifuge at 5,000 × g for 10 min to collect the bacteria;

[0065] 3. Discard the culture medium, add 500 μL Solution I / RNaseA mixture, and vortex to completely suspend the cells;

[0066] Note: Before using Solution I, please add RNase A according to the instructions and mix well.

[0067] 4. Transfer the solution to a new 2 mL centrifuge tube, add 500 μL Solution II, gently invert to mix, and place the mixture at room temperature for 2-3 minutes; the lysis time after adding Solution II should not exceed 5 minutes; seal and store.

[0068] 5. Add 250 μL pre-cooled N3 Buffer and gently invert the centrifuge tube several times until a white flocculent precipitate is formed;

[0069] 6. Centrifuge at room temperature and 13,000 × g for 10 min, and transfer the supernatant to a new 1.5 mL centrifuge tube;

[0070] 7. Add 0.1 times the volume of ETR Solution in the supernatant, mix by inverting 10 times, and place on ice for 10 minutes;

[0071] Note: After adding ETR Solution, the solution becomes turbid and becomes clear after ice bath;

[0072] 8. Incubate at 42°C for 5 min, the solution becomes turbid again;

[0073] 9. Centrifuge at 25°C and 12,000×g for 3 min, transfer the upper aqueous phase (containing DNA) to a new 1.5 mL centrifuge tube, add 0.5 times the volume of anhydrous ethanol, mix by inverting 6-7 times, and let stand at room temperature for 1-2 min;

[0074] 10. Transfer the mixture (up to 700 μL at a time) to a 2 mL collection tube. The DNA binding column was centrifuged at 10,000 × g for 1 min at room temperature, and the filtrate was discarded;

[0075] 11. Repeat step 10 until all the mixed solution has been transferred through the column;

[0076] 12. Put the column back into the collection tube, add 500μL HBC Buffer, centrifuge at maximum speed for 1min, and discard the filtrate; HBC Buffer must be diluted with isopropanol according to the instructions before use.

[0077] 13. Put the column back into the collection tube, add 700μL DNA Wash Buffer, centrifuge at maximum speed for 1min, and discard the filtrate DNA Wash Buffer. Before use, please add anhydrous ethanol to dilute it according to the instructions;

[0078] 14. Repeat step 13;

[0079] 15. Put the column back into the collection tube and centrifuge the empty column at 13,000 × g for 2 min to dry the column matrix;

[0080] 16. Place the column in a clean 1.5 mL centrifuge tube, add 80-100 μL Elution Buffer to the column matrix, let stand for 1 min, and centrifuge at maximum speed for 1 min to elute the DNA;

[0081] 17. Discard the column and store the DNA product at -20°C to obtain pLV3-TXNIP-56 plasmid and pLV3-TXNIP-231 plasmid;

[0082] (2) Amplification of U6 promoter-gRNA scaffold fragment

[0083] SnapGene software was used to design homologous recombination primer Multi-target-pLV3 (as shown in Table 5), and the U6 promoter-gRNA scaffold fragment on the pLV3-TXNIP-56 single target knockout vector was amplified by PCR. The amplification reaction system is shown in Table 6. The amplification reaction conditions are: pre-denaturation at 95°C for 5 min; then denaturation at 95°C for 30 s; then annealing at 60°C for 30 s; and then extension at 72°C for 30 s; a total of 39 cycles.

[0084] Table 5 Primer sequences

[0085]

[0086] Note: The lowercase letters are EcoR I restriction sites; the italic parts are the homologous sequences of the pLV3 vector.

[0087] Table 6 Amplification system

[0088]

[0089]

[0090] (3) Enzyme digestion of TXNIP gene knockout single target vector

[0091] The pLV3-TXNIP-231 plasmid was digested with EcoR I restriction endonuclease. The digestion system is shown in Table 7. The reaction conditions are: incubation at 37°C for 30 min, followed by treatment at 65°C for 20 min to inactivate the enzyme.

[0092] Table 7 Enzyme digestion system

[0093] Reagents Dosage pLV3-TXNIP-231 plasmid 1 μg EcoRI 1μL rcutsmart 5μL <![CDATA[ddH2O]]> Add to 50 μL

[0094] (4) Connection

[0095] The U6 promoter-gRNA scaffold fragment was connected to the extracted pLV3-TXNIP-231 plasmid using the Seamless Cloning Kit. The connection system is shown in Table 8. The reaction conditions are: incubation at 50°C for 15 minutes to obtain a connection product.

[0096] Table 8 Connection system

[0097] Reagents Dosage U6 promoter-gRNA scaffold fragment 100ng Linearize pLV3-TXNIP-231 plasmid 60ng 2×Seamless Cloningmix 10μL <![CDATA[ddH2O]]> Add to 20 μL

[0098] (5) Conversion

[0099] The ligation product obtained in (4) is transferred into competent cells. The specific steps are as follows:

[0100] ① Place 100 μL TOP10 competent cells in a 1.5 mL EP tube, add 10 μL ligation product, pipette up and down to mix, and place on ice for 30 min.

[0101] ② Place the EP tube in a 42°C water bath for 90 seconds, then quickly transfer to ice and cool for 2-3 minutes.

[0102] ③ Add 500 μL of LB medium without ampicillin to the EP tube, mix well, and culture on a shaker at 37°C and 200 rpm / min for 45 min.

[0103] ④ Pipette 40 μL of transformed TOP10 competent cells and spread them on LB solid medium containing ampicillin.

[0104] ⑤Use a spreader to spread the cells evenly and place the plate in a 37°C incubator for 16 hours.

[0105] ⑥ Pick a single colony and culture it in 30 mL of liquid culture medium containing 100 ng / mL ampicillin at 37°C and 200 rpm / min for 16 h, then centrifuge it at 4500 rpm for 10 min, discard most of the culture medium, resuspend the bacterial pellet, preserve the bacteria, and send the bacterial solution to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results are as follows Figure 3 shown.

[0106] like Figure 3 As shown, the obtained recombinant plasmid was clone-positive, and the recombinant plasmid contained two complete U6 promoter-sgRNA-gRNA scaffold sequences, and both sgRNA sequences were correct. This result shows that the present invention successfully constructed a TXNIP gene dual-target expression plasmid.

[0107] (III) Performance testing

[0108] 1. TXNIP gene knockout efficiency detection

[0109] The pLV3-NC (empty lentiviral vector) and TXNIP-KO (double-target knockout vector for TXNIP gene) plasmids were transfected into bovine mammary epithelial cells by liposome method. Total protein of cells was extracted 48 hours after transfection, and the protein expression level of TXNIP gene was detected by Western blot method. The results are shown in Figure 2. Figure 4 shown.

[0110] Depend on Figure 4It can be seen that the protein of the TXNIP gene in the bovine mammary epithelial cells transfected with the bovine TXNIP gene dual-target vector is almost not expressed, and the difference is extremely significant compared with the control group (P<0.01), which can be used for subsequent experiments. The results show that the TXNIP gene dual-target expression plasmid of the present application reduces the risk of off-target effects, has a high knockout efficiency, and can be used for the study of the function of the bovine TXNIP gene.

[0111] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A bovine TXNIP gene dual-target knockout vector, characterized in that: It includes a lentiviral expression vector, a first sgRNA sequence targeting a bovine TXNIP coding region connected to the lentiviral expression vector, and a second sgRNA sequence targeting a TXNIP coding region connected to the lentiviral expression vector, wherein the nucleotide sequences of the upstream primer and the downstream primer corresponding to the first sgRNA sequence are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively, and the nucleotide sequences of the upstream primer and the downstream primer corresponding to the second sgRNA sequence are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

2. The bovine TXNIP gene dual-target knockout vector according to claim 1, characterized in that: The first sgRNA sequence targets the sgRNA56 site in the bovine TXNIP coding region.

3. The bovine TXNIP gene dual-target knockout vector according to claim 1, characterized in that: The second sgRNA sequence targets the sgRNA231 site in the bovine TXNIP coding region.

4. The bovine TXNIP gene dual-target knockout vector according to claim 1, characterized in that: The lentiviral expression vector is selected from a lentiviral expression vector.

5. The bovine TXNIP gene dual-target knockout vector according to claim 4, characterized in that: The lentiviral expression vector is selected from pLV3-U6-MCS-sgRNA-Cas9-EGFP-Puro (pLV3).

6. The method for constructing the bovine TXNIP gene dual-target knockout vector according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. Annealing the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2 to obtain a first annealing product; S2. Annealing the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain a second annealing product; S3. connecting the first annealing product into a lentiviral expression vector linearized after being digested with restriction endonuclease Esp3I to obtain a first single target knockout vector; Amplifying the first single target knockout vector and the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6 to obtain an amplified product; S4. connecting the second annealing product into a lentiviral expression vector linearized after being digested with restriction endonuclease Esp3I to obtain a second single target knockout vector; Digesting the second single target knockout vector with restriction endonuclease EcoR I to obtain a digestion product; S5. Connect the amplified product and the enzyme-digested product to obtain the bovine TXNIP gene double-target knockout vector.

7. The method for constructing the bovine TXNIP gene dual-target knockout vector according to claim 6, characterized in that: In step S1, the annealing includes: treating at 95°C for 2 minutes, then decreasing by 0.1°C every 8 seconds to 25°C for about 90 minutes, and temporarily storing at 4°C; and / or, in step S1, the molar ratio of the nucleotide sequence of SEQ ID NO.1 to the nucleotide sequence of SEQ ID NO.2 is 1:1; And / or, in step S2, the annealing comprises: treating at 95°C for 2 minutes, then decreasing by 0.1°C every 8 seconds to 25°C for about 90 minutes, and temporarily storing at 4°C; And / or, in step S2, the molar ratio of the nucleotide sequence shown in SEQ ID NO.3 to the nucleotide sequence shown in SEQ ID NO.4 is 1:

1.

8. The method for constructing the bovine TXNIP gene dual-target knockout vector according to claim 6, characterized in that: In step S3, the molar ratio of the first annealing product to the linearized lentiviral expression vector is 3-10:1; And / or, in step S5, the mass ratio of the amplification product to the enzyme cleavage product is 50-100:50-100.

9. A cell line, characterized in that The bovine TXNIP gene dual-target knockout vector according to any one of claims 1 to 5 and / or the bovine TXNIP gene dual-target knockout vector constructed by the method according to any one of claims 6 to 8 is co-transfected with pMD2.G and pSPAX2 plasmids to package 293FT cells for lentivirus. After 48 hours, the viral supernatant is collected and infected with bovine mammary epithelial cells, and the cells are screened with puromycin to obtain the lentivirus.

10. Use of the bovine TXNIP gene dual-target knockout vector according to any one of claims 1 to 5 and / or the bovine TXNIP gene dual-target knockout vector constructed according to the method according to any one of claims 6 to 8 and / or the cell line according to claim 9 in the study of bovine TXNIP function.

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