CN107058302B - Targeted Knockout of Nrf1β Gene Recognition Sequence Pair, Talens, Vector Pair and Application in Human Hepatocytes - Google Patents

本发明提供人源肝细胞中Nrf1β基因定向敲除识别序列对、Talens、载体对及应用，所述识别序列对由左臂识别序列和右臂识别序列组成，左臂识别序列的核苷酸序列如SEQ ID NO.1所示，右臂识别序列的核苷酸序列如SEQ ID NO.2所示；所述核酸酶Talens由识别SEQ ID NO.1核苷酸序列的核酸酶左臂Nrf1β‑Talen L和识别SEQ ID NO.2核苷酸序列的核酸酶右臂Nrf1β‑Talen R组成；所述载体对由Nrf1β‑Talen L载体和Nrf1β‑Talen R载体组成；所述载体对能在定向敲除人源肝细胞系中Nrf1β基因、构建Nrf1β基因敲除型人源肝细胞系中应用。The present invention provides a pair of recognition sequences for directional knockout of Nrf1β gene in human hepatocytes, a Talens, a pair of vectors, and applications, wherein the pair of recognition sequences consists of a left arm recognition sequence and a right arm recognition sequence, and the nucleotide sequence of the left arm recognition sequence As shown in SEQ ID NO.1, the nucleotide sequence of the right arm recognition sequence is shown in SEQ ID NO.2; the nuclease Talens is composed of the nuclease left arm Nrf1β- which recognizes the nucleotide sequence of SEQ ID NO.1 Talen L and the right arm Nrf1β-Talen R of the nuclease recognizing the nucleotide sequence of SEQ ID NO. In addition to the Nrf1β gene in the human hepatocyte cell line, it is applied in the construction of the Nrf1β gene knockout human hepatocyte cell line.

Nrf1 β gene targeted knockout recognition sequence pair, Talens, vector pair and application in human hepatocytes

Technical Field

The invention belongs to the technical field of gene knockout, and particularly relates to a targeted knockout recognition sequence pair, Talens, a vector pair and application of Nrf1 β gene in human hepatocytes.

Background

However, as the Nrf1 protein in cells has a plurality of subtypes including Nrf1 α, Nrf1 β, Nrf1 chi, Nrf1D and the like and a plurality of post-translational modifications, the function of Nrf1 cannot be thoroughly researched.

In the traditional research method, the principle of researching the function of the Nrf1 by using the siRNA interference technology is that the mRNA of the Nrf1 in a cell is degraded, so that the level of the Nrf1 protein in the cell is reduced, and the Nrf1 in the cell cannot be completely eliminated, so that the obtained research result cannot eliminate the influence of the residual Nrf1 protein, in addition, the siRNA technology also has the off-target problem, and the stability and the repeatability of the siRNA technology are poor; the research on the Nrf1 by using the homologous recombination system gene knock-out technology is to knock out all Nrf1 genes on a genome, but the method is complicated to operate, long knock-out fragments on the genome can cause uncontrolled transcription, and the functions of different Nrf1 subunits cannot be distinguished.

How to make research of the Nrf1 become convenient and fast, all Nrf1 can be knocked out without changing genome into genetic background by making larger length change, and research results are stable, reliable and repeatable, and the problem to be solved in the research field of the Nrf1 is urgent.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to solve the technical problems of how to provide a targeted knockout recognition sequence pair, Talens, a vector pair and application of the Nrf1 β gene in the human hepatocyte, so that the method is convenient and quick to operate, the Nrf1 β gene is completely knocked out under the genetic background that the genome is not changed in large length, and the Nr f1 β protein is not remained in the hepatocyte.

In order to solve the technical problems, the technical scheme includes that an identification sequence pair is directionally knocked out by Nrf1 β gene in a human hepatocyte based on Talen, the identification sequence pair is composed of a left arm identification sequence and a right arm identification sequence, the nucleotide sequence of the left arm identification sequence is shown as SEQ ID No.1, and the nucleotide sequence of the right arm identification sequence is shown as SEQ ID No. 2.

The transcription activation-like effector nuclease Talens is used for directionally knocking out Nrf1 β genes in human hepatocytes and consists of a nuclease left arm Nrf1 β -Talen L for identifying a nucleotide sequence of SEQ ID NO.1 and a nuclease right arm Nrf1 β -Talen R for identifying a nucleotide sequence of SEQ ID NO.2, wherein the Nrf1 β -Talen L is formed by fusing a Tale-L for identifying a nucleotide sequence of SEQ ID NO.1 and a non-specific endonuclease Fokl, the Nrf1 β -Talen R is formed by fusing a Tale-R for identifying a nucleotide sequence of SEQ ID NO.2 and a non-specific endonuclease Fokl, the amino acid sequence of the Tale-L is shown in SEQ ID NO.3, and the amino acid sequence of the Tale-R is shown in SEQ ID NO. 4.

The vector pair for directionally knocking out Nrf1 β genes in human hepatocytes comprises an Nrf1 β -Talen L vector and an Nrf1 β -Talen R vector, wherein the Nrf1 β -Talen L vector comprises a nucleotide sequence for coding Tale-L, the Nrf1 β -Talen R vector comprises a nucleotide sequence for coding Tale-R, and the Nrf1 β -Talen L vector and the Nrf1 β -Talen R vector both have puromycin resistance genes.

Further, the Nrf1 β -Talen L vector is a TALEN-LEFT vector containing a nucleotide sequence for coding the Tale-L in claim 2, and the Nrf1 β -Talen R vector is a TALE-RIGHT vector containing a nucleotide sequence for coding the Tale-R in claim 2.

The vector for directionally knocking out the Nrf1 β gene in the human hepatocyte is applied to directionally knocking out the Nrf1 β gene in the human hepatocyte and constructing a Nrf1 β gene knock-out human hepatocyte line.

Furthermore, the human hepatocyte is human hepatocyte HL7702 cell.

The construction method of the Nrf1 β gene knockout human-derived liver cell line comprises the following steps:

1) transfecting the vector pair into human hepatocytes by a transfection method;

2) screening the cultured human hepatocytes subjected to transfection in the step 1) by puromycin to obtain human hepatocytes successfully transfected with Nrf1 β -TalenL vector plasmids and Nrf1 β -TalenR vector plasmids;

3) digesting and suspending the human hepatocytes successfully screened in the step 2) and transfected with the Nrf1 β -Talen L vector plasmid and the Nrf1 β -Talen R vector plasmid by using pancreatin, continuously culturing until single cells are cloned and grown, selecting a community grown by the single cells and having a good cell state, and continuously expanding and culturing to obtain the Nrf1 β gene knockout type human hepatocyte line.

Further, the specific steps of transfecting the vector pair into the human hepatocytes by a transfection method are as follows:

A. adding human-derived hepatocytes into each hole of a cell culture pore plate, culturing the human-derived hepatocytes by adopting a DMEM (DMEM) culture medium until the human-derived hepatocytes grow and cover 80% of the bottom of the pore plate, sucking out the DMEM culture medium in each hole, adding Opti-MEM, and dividing each hole of the cell culture pore plate into a transfection group and a control group for later use;

D. after the completion of the culture, the liquid in the transfection group and the control group is sucked out, and DMEM medium containing 10wt.% FBS is added to the wells of the transfection group and the control group, respectively, to complete the transfection step.

Further, the puromycin screening step 1) of the transfection of the cultured human hepatocytes by the puromycin in the step 2) specifically comprises the steps of adding puromycin into a DMEM medium containing 10wt.% of FBS, uniformly mixing to obtain a puromycin-containing medium, wherein the final concentration of puromycin in the puromycin-containing medium is 2 mug/mL, continuously culturing the human hepatocytes in a control group by using the puromycin-containing medium after the human hepatocytes are transfected with Nrf1 β -Talen L carrier plasmid and Nrf1 β -Talen R carrier plasmid for 48h, and obtaining the human hepatocytes which are successfully transfected with the Nrf1 β -Talen L carrier plasmid and Nrf1 β -Talen R carrier plasmid as the human hepatocytes which are still alive in the A hole.

Furthermore, the human hepatocyte is human hepatocyte HL7702 cell.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention utilizes TALEN technology to make gene editing simple and easy, can edit any nucleic acid base site, realizes gene editing by knocking out bases with non-3 times to form coding mutation, has only insertion or deletion of a plurality of bases on a genome, but can completely eliminate the expression of a target gene on the protein level, concretely, the invention utilizes Talen technology to edit Nrf1 genome base sequence in human hepatocyte HL7702 cell, and cultures and identifies successfully to manufacture a stable monoclonal cell line 7B2 knocked out by Nrf1 β through screening of monoclonal cell lines, the CDS region of Nrf1 of the monoclonal cell line is deleted for a plurality of bases, so that the Nrf1 protein is completely knocked out on the protein level due to the code shift deletion of the coding region bases, thereby realizing the extremely small change of the genome to achieve the complete knocking out of the Nrf1 β protein and obtaining excellent gene editing effect.

2. The 7B2 cell line subjected to gene knockout is a unicellular clone cell line obtained by a monoclone culture and selection technology, and the obtained 7B2 cell line is found to have excellent and stable state and form after being cultured for several months, which indicates that the gene knockout cell line obtained by the invention has good stability.

3. The 7B2 is a cell line with completely knocked-out Nrf1 β protein, and the obtained research result can completely show the function of Nrf1 β in hepatocytes and can be used as an excellent material for the function research of Nrf1 β in hepatocytes, the 7B2 cell line is a cell line with knocked-out Nrf1 protein beta subtype, and the cell line can be used together with a cell line with knocked-out other subtypes of Nrf1 protein to realize the research on the functions of the Nrf1 protein subtypes.

4. The 7B2 cell line constructed by the method is a monoclonal cell line, the genetic expression is stable, the repeatability of the obtained research result is good, and the research result is accurate and reliable.

Drawings

FIG. 1 is a diagram of Nrf1 β gene editing site selection and TALEN recognition site selection;

FIG. 2 is a diagram of an alignment of Nrf1 genome Nrf1-7B2-1, Nrf1-7B2-2 with normal Nrf1 genome Nrf1-WT in 7B 2;

FIG. 3 is a diagram showing the sequencing result of the knock-out site sequence amplified by PCR using the 7B2 genome as a template;

FIG. 4 is a graph showing the quantitative PCR detection of the mRNA level changes of Nrf1 in 7B2 and HL7702 cells;

FIG. 5 is a diagram of detection of Nrf1 protein in 7B2 and HL7702 cells by Western blotting.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The present invention is implemented on the premise of the technology of the present invention, and the detailed embodiment and the specific operation process are given to illustrate the inventive aspects of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1 selection of editing sites and design and construction of Nrf1 β -TALEN plasmid

Designing Nrf1 β knock-out site PCR reaction amplification primer sequences:

the Nrf1 β F primer was inserted into the Kpn I restriction endonuclease cut site and the Nrf1 β R primer was inserted into the Bgl II restriction endonuclease cut site (base marked in bold is the corresponding restriction endonuclease recognition site.)

Nucleotide sequences of Nrf1 β -Talen L and Nrf1 β -Talen R are respectively inserted into a TALEN-LEFT (containing puromycin resistance gene) and a TALE-RIGHT expression vector according to a TALEN plasmid kit using instruction, plasmids are extracted, and Nrf1 β -Talen L vector plasmid and Nrf1 β -Talen R vector plasmid are respectively obtained.

Example 2 screening, culture and characterization of Nrf1 β Gene knock-out monoclonal cell lines

1) Nrf1 β -TALEN plasmid transfected HL7702 cells:

a. 2 holes of a six-hole plate of HL7702 cell species are respectively marked as A (plasmid transfection group) and B (control group), 30 ten thousand cells in each hole are cultured overnight by adopting a DMEM medium, and Nrf1 β -TALEN plasmid is prepared to be transfected when the cells grow to about 80% of a full bottom plate at the bottom of the hole plate.

b. The A, B wells were aspirated in DMEM medium, and 800. mu.L of Opti-MEM was added.

liquid 2: 100 μ L of Opti-MEM;

liquid 3: adding 200 mu L of Opti-MEM into lipo200018 mu L, uniformly mixing and standing for 5 minutes;

f. putting the hole plate added with the liquid in the steps d and e into an incubator, and adding CO with the volume concentration of 5 percent₂After 8 hours of incubation at 37 ℃, the medium was aspirated from each well, and 2mL of DMEM medium containing 10wt.% FBS was added to each of the A, B wells.

2) Puromycin screening resulted in successful transfection of cells:

preparing a puromycin-containing culture medium: puromycin was added to DMEM medium containing 10wt.% FBS and mixed well to a final puromycin concentration of 2 μ g/mL.

After the HL7702 cells in the step 1) are transfected with the Nrf1 β -TALEN plasmid for 48 hours, the cells in A, B wells are continuously cultured by using a puromycin-containing culture medium, and the volume concentration of the CO is 5 percent₂And culturing at 37 ℃ for 3-5 days until all cells in the B hole of the control group die, wherein the rest cells in the A hole are HL7702 cells which are successfully transfected with the Nrf1 β -TALEN plasmid.

3) Selecting a monoclonal cell line:

digesting and suspending HL7702 cells successfully transfected with the Nrf1 β -TALEN plasmid obtained in the step 2) by pancreatin, and specifically, washing the cells in the culture plate for 2 times by using a serum-free DMEM medium, removing serum residues in the original culture medium, sucking the culture medium, adding 2mL of pancreatin, and digesting in an incubator at 37 ℃ for 3 to 5 minutes.

After digestion, the digested cells were counted using a hemocytometer, and 100 cells were aspirated according to cell concentration and added to 10mL of DMEM medium containing 10wt.% FBS and mixed well.

Placing the mixed 96-well plate containing cell culture medium seeds into an incubator at a volume concentration of 5% CO, wherein the inoculation amount of each well is 100 mu L₂And cultured at 37 ℃ until single cell clone grows.

And observing the cell colony in the 96-well plate under a microscope, and selecting the colony which grows from the single cell and has a good cell state to continue expanding and culturing to obtain the monoclonal cell line.

4) And (5) identifying a monoclonal cell line.

Extracting genome, total RNA and total protein from the monoclonal cell line which is subjected to the enlarged culture in the step 3) and a control cell line (HL 7702).

A monoclonal cell line genome is used for PCR, primers Nrf1 β F and Nrf1 β R are used for amplifying a Nrf1 β -TALEN target sequence fragment and sequencing, and the base editing effect of the Nrf1 gene is detected.

Total RNA from the monoclonal cell line was reverse transcribed to cDNA and the mRNA level changes of Nrf1 in the monoclonal cell line were detected by quantitative PCR techniques.

Changes in the Nrf1 protein level in the total protein of the monoclonal cell line were detected by western blotting technique using Nrf1 antibody.

Through the above 3 detection methods, a monoclonal cell line 7B2 in which Nrf1 β in HL7702 cells was successfully knocked out was identified, and the identification results showed that Nrf1 gene in 7B2 cell line lacked 5/12 DNA bases (5 bases lacked on one chromosome, 12 bases lacked on the other chromosome, all disrupted initiation codon) (fig. 2, fig. 3), Nrf1 in 7B2 cell line did not significantly change in expression of mRNA level (fig. 4), Nrf1 β protein band in 7B2 cell line completely disappeared (fig. 5), the results showed that monoclonal cell line 7B2 in which Nrf1 β protein in human hepatocyte HL7702 was completely knocked out was successfully obtained.

When subculturing the obtained 7B2 cell line, the cell line was cultured in a high-sugar DMEM medium containing diabody and 10% FBS in a 5% CO atmosphere₂At 37 ℃. The culture medium is replaced every 2-3 days, and the cells are spread on the bottom of the culture bottle for passage. When the cell line is passaged, adherent cells are washed by PBS buffer solution, then trypsinized for 5-10 minutes, a high-sugar DMEM medium containing 10% FBS is added to stop digestion, the cells are blown out by a pipette, and the cells are transferred into 2 bottles or 3 bottles.

The above examples of the present invention are merely illustrative of the present invention and are not intended to limit the embodiments of the present invention. Variations and modifications in other variations will occur to those skilled in the art upon reading the foregoing description. Not all embodiments are exhaustive. All obvious changes and modifications of the present invention are within the scope of the present invention.

SEQUENCE LISTING

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