CN108486154A - A kind of construction method of sialidase gene knock-out mice model and its application - Google Patents

Abstract

The present invention relates to the field of biotechnology, and provides a method for constructing a sialidase gene knockout mouse model and its application, using a recombinant CRISPR knockout plasmid vector targeting the mouse genome NPL gene in vivo and in vitro, through fertilized egg microscopy The injection technique successfully established a diploid double knockout NPL mouse knockout model. NPL plays an important role in the pathophysiological process of the body. The malignant transformation of tumor cells is often accompanied by the overexpression of sialic acid. Now it has become one of the important markers of malignant transformation and metastasis of tumors. This model builds a new research platform for the study of the process of cell carcinogenesis, which is conducive to the study of key steps in tumor formation and metastasis, and provides a basis for the development of new anti-tumor drugs.

Description

A method for constructing a sialidase gene knockout mouse model and its application

technical field

The invention belongs to the technical field of gene editing, relates to molecular biology, cell biology, genetics and other research methods, in particular to a method for constructing a sialidase gene knockout mouse model and its application.

Background technique

The establishment of gene knockout mouse models has created the possibility for precise research on the relationship between genes and human diseases. Since the birth of genetically modified animals, people have paid great attention to them. With continuous development and improvement in recent years, genetically modified animals have gradually begun to play an extremely important role in many fields. For the entire society, these genetically modified animals Animals have greatly promoted the development of social economy and scientific research. Constructing transgenic animal models, especially gene knockout and knockin animal models, is one of the most intuitive and reliable ways to study gene function, especially opening up a new platform and direction for the study of tumor occurrence and development. Knockout methods for gene knockout mice have been developed so far, mainly including ES cell targeting technology, TALEN technology, and Cre/loxP conditional knockout methods. With the development of CRISPR/Cas9 technology, because of its strong targeting, Due to the advantages of high knockout efficiency and convenient operation, it is very suitable for the preparation of target gene knockout mice.

Microinjection technology, microinjection is one of the methods developed earlier for introducing foreign genes into cells to construct transgenic animals, but if foreign DNA fragments are integrated into the host genome through non-homologous recombination, their integration Very random and less efficient. The combination of CRISPR/Cas9 technology and microinjection technology can greatly solve the problem of the long production cycle of knockout mice, and at the same time realize the need for precise gene editing of fertilized eggs and embryos.

Sialic acid has a great relationship with tumorigenesis and progression. Studies have shown that malignant transformation of cells is often accompanied by overexpression of sialic acid. Effectively constructing a stable and completely knockout mouse of the NPL gene will greatly help to explore the key role of the NPL gene and its related pathways in tumor adhesion and metastasis, and promote the discovery of NPL-related interaction genes, which will contribute to the development of tumor progression and metastasis. A new research platform has been set up for the research and related drug screening.

Contents of the invention

The present invention aims to provide a method for constructing a sialidase gene knockout mouse model and its application. Including the verification of effective targeting of NPL gene sgRNA recombination knockout CRISPR plasmid vector in mouse-derived cells, microinjection of recombinant targeting NPL gene CRISPR system into fertilized eggs, and reinfusion into oviduct to obtain newborn mice, and verification of NPL by mouse tail sequencing Knockout positive mice.

In order to realize the described purpose of the present invention, adopt following experimental technical scheme:

The knockout plasmid capable of expressing the CRISPR/Cas9 gene editing system was selected as Px459 vector, the sgRNA targeting the NPL gene was designed, and the recombinant Px459 plasmid was obtained by enzyme digestion and ligation.

The construction method of the above-mentioned recombinant ICAM-1 knockout recombinant Px459 plasmid vector, the following steps:

1) According to the CDS region sequence of the NPL gene (Chromosome 1 - NC_000067.6), design the sgRNA targeting the NPL gene to obtain the NPL-sgRNA with the sequence GGTGTCCGCGGCGGAATCCG;

2) Synthesize NPL-sgRNA forward and reverse oligonucleotide sequences, add BbsI enzyme cutting sites at both ends; form double-stranded oligonucleotides by gradient annealing;

NPL-sgRNA forward and reverse oligonucleotide sequences are:

Positive strand F: CACCGGGTGTCCGCGGCGGAATCCG;

Anti-chain R:AAACCCGGATTCCGCCGCGGACACC;

3) Recover the Px459 plasmid vector after digestion with BbsI, and connect it with double-stranded NPL-gRNA by T4 ligase to form a recombinant gene knockout vector; single-clonal picking and sequencing of transformed competent strains to obtain the required NPL-gRNA/Cas9 recombination Knockout CRISPR vector, i.e. recombinant Px459 plasmid.

A method for knocking out the NPL gene in mouse fibroblast L929 cells and verifying the knockout, the specific operations are as follows:

1) Using lipo 3000, the mass ratio of plasmid to lipo is 1:2.5, and the recombinant knockout plasmid obtained above is transfected into L929 cells in a six-well plate. After 24h, puromycin antibiotics were used for screening.

2) Continuous drug screening for three days, replace with normal culture medium for culture, and wait for a new single cell mass to grow.

3) Digest the monoclonal cell pellet with trypsin and resuspend the cells. After adhering to the wall, keep changing the liquid regularly. Ensure the proliferation and growth of monoclonal cells, and extract the cell genome.

4) Use the synthetic verification primer PCR to recover the DNA of the target fragment through gel recovery, sequence and compare the wild-type genome, and detect the genetic modification of the sgRNA sequence.

5) If there are double peaks in the sequencing profile, or there are base changes in the sgRNA sequence region (see Figure 1). It is a biologically active sgRNA.

A method for establishing a microinjection, the specific operations are as follows:

1) Surgically obtain fertilized eggs from successfully fertilized female mice.

2) Using a motorized microinjector, inject the CRISPR plasmid targeting the NPL gene into fertilized eggs.

3) The fertilized eggs that have been successfully injected with the plasmid are transferred to the fallopian tube embryos. After suturing, the mice were allowed to wake up.

The method of positive knockout mouse verification, the specific method is as follows:

1) At least one week after the birth of the mouse, the tip of the mouse tail can be cut off.

2) Rat tail grinding was used to extract the genome.

3) Use the designed verification primers for PCR amplification, and recover the target fragment containing the sgRNA sequence.

4) Sequencing was compared with the wild-type genome, and T-A cloning was used to analyze the sequence changes of diploid double copies (see Figure 2).

The advantages of the present invention are:

1. Combining CRISPR/Cas9 technology with microinjection technology for the construction of knockout mice is faster and more efficient.

2. The experiment period is short, which saves the experiment cost.

3. NPL knockout mice are used in the study of tumor formation and progression, and the effect is more direct and significant. The related genes of NPL make its real-time monitoring in vivo possible.

Description of drawings

Figure 1 L929 cell level sequencing verification double peak and sequence alignment.

Figure 2 Sequence comparison of mouse tail sequencing verification and T-A cloning double-copy knockout verification diagram.

Figure 3 Comparison of subcutaneous implanted tumors between NPL knockout mice and wild-type mice.

Detailed ways

Example 1 Construction of Recombinant NPL Knockout CRISPR System Plasmid

(1) Design of sgRNA

First, find the genome sequence of the mouse NPL gene through NCBI, and design the sgRNA targeting the NPL gene (sequence is GGTGTCCGCGGCGGAATCCG) on the sgRNA design website of Zhang Feng’s research group http://crispr.mit.edu/, and reverse complementation to obtain the reverse strand sequence. Residues after digestion of the BbsI restriction site were added to both ends, and synthesized.

The two fragments after synthesis are as follows:

F: CACCG GGTGTCCGCGGCGGAATCCG;

R: AAAC CGGATTCCGCCGCGGACACC C;

(2) sgRNA annealing,

Annealing reaction system:

Mix the prepared liquid evenly, centrifuge briefly and place in a gradient PCR instrument, run the program: 95°C, 10min; 85°C-65°C decreases by 0.1°C per second, 25°C, to form a double-stranded oligonucleotide;

Positive strand F: CACCGGGTGTCCGCGGCGGAATCCG;

Anti-chain R: AAACCCGGATTCCGCCGCGGACACC.

(3) Digest with BbsI

Reaction system for restriction endonuclease BbsI to digest Px459 vector:

Gently mix and incubate at 37°C for 10 minutes to connect.

(4) Carrier and sgRNA connection

T4 DNA Ligase kit reaction system:

Mix well, PCR machine program: 16 ℃, 3 h; 65 ℃, 10 min; 4 ℃, ∞.

(5) Transformation of DH5α competent cells

Competent cells 50 μL + recombinant plasmid 3 μL, mix well and add to 1.5 mL EP tube; ice bath for 30 min; 42 °C water bath for 45 s; ice bath for 3 min; add 800 μL LB; For the supernatant, keep about 80ul to resuspend and mix evenly to smear the plate; seal and invert overnight at 37°C.

(6) Sequencing and conservation

Three single clones were picked from each plate for shaking bacteria, and the bacteria liquid was taken for sequencing; the sequence comparison of the sequencing results showed that the vector recombination was successfully constructed, and the plasmid was extracted for subsequent cell and animal experiments.

Example 2 Verification of the knockout vector at the cellular level

Before using the CRISPR system to knock out the vector for the preparation of knockout mice, it is necessary to verify whether the designed sgRNA is effective, and the knockout verification can be performed in the mouse cell line. Proceed as follows:

cell transfection

1) Take the cells in the logarithmic growth phase and inoculate them in a six-well plate. Establish the experimental group and the control group, and wait for the cell plating rate to reach 75-80%.

2) Take two EP tubes, add MEM (50 μl) + lipo3000 (3.75 μl) to tube I, add MEM (50 μl) + DNA (2.5 μg) + P3000 (5 μl) to tube II.

3) Add the liquid in II to I, mix well and let it stand for 5 minutes, then add it to a six-well plate and culture it in a cell culture incubator.

Genome Extraction and Validation

Primers were designed to amplify about 300 bp at the upper and lower ends of the sgRNA site of the NPL gene, for a total of about 600 bp fragments; the amplified fragments were sequenced and compared. Compared with the wild-type genome sequence, if there is a change in the sgRNA site, it means that the designed CRISPR recombinant vector has biological activity, otherwise it is invalid.

The primer sequence is: Forward: GAGTGACGTGCGCGCTTTTA;

Reverse: TGATGAGCGGGAGCGGA;

The PCR reaction system is as follows:

PCR conditions: 98°C 3min——[98°C 10s, 55°C 30s, 72°C 1min]×35 cycles——72°C 7min——4°C ∞

Example 3

Fertilized egg retrieval

(1) Egg donor female mice were intraperitoneally injected with 0.1ml (5U) of pregnant horse serum gonadotropin PMSG, thereby promoting the maturation of female mice's eggs.

(2) After the injection, the recipient female mouse and the ligated male mouse were caged together, and they were placed in a pseudo-pregnant state through normal mating;

(3) After pseudopregnancy, female mice were injected with human chorionic gonadotropin hCG 0.1ml (5U), and then mated with normal male mice at a ratio of 1:1.

(4) On the second day, the donor mice and recipient mice were checked for pregnancy plugs, and the mice with pregnancy plugs were used for fertilized egg collection.

The specific operation of microinjection is as follows:

(1) Use the egg transfer device to absorb the fertilized egg cells and place them in the M2 medium on the glass slide;

(2) Absorb and fix the prepared fertilized eggs with a fixed needle, insert the needle into the cytoplasm, quickly inject about 1 pl of injection solution using a syringe pump, and at the same time quickly withdraw the injection needle, and transfer the injected fertilized egg cells to a clean area ;

(3) After the injection, observe the changes of the fertilized eggs. If the cytoplasm of the fertilized eggs leaks out or the morphology of the fertilized eggs is damaged, the injection fails and a new injection is required.

(4) Collect the fertilized eggs that have been successfully injected and transfer them to an organ culture dish containing fresh M16 culture medium, and place them in a carbon dioxide incubator for culture.

Intra-fallopian embryo transfer

(1) Place the recipient mother mouse on its side after anesthesia and place it under the microscope. After 70% alcohol disinfection, cut a small incision on the skin with scissors from the last rib along the dorsal midline, and through a dorsal incision perpendicular to the spine, you can access the fallopian tubes and ovaries on both sides. Slide the skin around so that the ovaries (orange) or fat pads (white) can be seen through the body wall. Then use clock forceps to pick up the body wall and cut a small opening where the ovary is.

(2) Before aspirating the embryos, first inhale a small amount of M2 culture solution in the transfer tube, then inhale a small air bubble, then inhale the M2 culture solution, and then inhale the second small air bubble, and repeat until the siphon effect can be reduced well Control the entry and exit of liquids.

(3) Hold the fat pad with forceps and pull out the attached left ovary, fallopian tube and uterus. The fat pad is clamped with small spring clips to expose the ovaries and fallopian tubes in view. Under a microscope, adjust the position of the oviduct of the recipient mother mouse so that the transplanted tube can be inserted into the oviduct. Use soft clips to pull out the ovary and fix it outside the body, and use pointed forceps to tear a small opening on the ovarian sac of the fimbria of the fallopian tube to find the fimbria of the fallopian tube. Carefully hold the umbrella of the fallopian tube with forceps, insert the transfer tube into the opening of the fallopian tube, and then blow the embryo and air bubbles into the ampulla of the fallopian tube.

(4) Release the small spring clips, pick up the fat pad with blunt forceps, and put the uterus, fallopian tubes, ovaries, etc. back into the body cavity. The body wall is sutured first, and then the skin wound is sutured. If necessary, repeat the above steps, the embryo can be transferred to the right fallopian tube. After the operation, the mice were placed in a clean mouse cage, and the mouse cage was placed on a heating pad at 37°C until it woke up.

Identification of NPL knockout mice

At least one week after the birth of the mouse, the tail tip of the mouse can be cut for genome extraction for PCR amplification and sequencing. The operation is the same as the cell-level genome verification method.

Differences in tumorigenesis and progression between knockout mice and wild-type mice

1) Cell preparation: Take human lung cancer A549 cells as an example. Routine digestion, centrifugation, HBSS-/-washing, HBSS-/- and Matrigel were mixed in equal proportions to make a cell suspension.

2) The mouse was anesthetized with an animal anesthesia apparatus, the hair on the right hind limb was shaved, and the cell suspension was slowly injected subcutaneously.

3) Sterilize with 70% alcohol and put it on a 37°C heating plate or other equipment until the mouse wakes up.

After 30 days, the difference in tumor size between wild-type mice and NPL knockout mice was observed. The tumor volume of NPL knockout mice was 2.16 cm ³ , and that of wild-type mice was 5.37 cm ³ . It can be found that the tumors of NPL knockout mice are smaller than those of wild-type mice (as shown in Figure 3), which verifies that NPL plays an important role in tumorigenesis. Therefore, NPL knockout mice can be used as a model for studying tumorigenesis and progression, and provide convenience for the screening of related drugs.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

SEQUENCE LISTING

<110> Fuzhou University

<120> A method for constructing a sialidase gene knockout mouse model and its application

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<170> PatentIn version 3.3

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Claims (6)

1. A method for constructing a sialidase gene knockout mouse model, characterized in that, based on the CRISPR/Cas9 technology, select the Px459 vector, design the sgRNA targeting the NPL gene, obtain the recombinant Px459 plasmid by restriction enzyme digestion, and then recombine The plasmid was injected into fertilized eggs to obtain sialidase gene knockout mice. 2. the construction method of a kind of sialidase gene knockout mouse model according to claim 1, is characterized in that, the construction method of described recombinant Px459 plasmid is: 1) According to the CDS region sequence of NPL gene, design sgRNA targeting NPL gene to obtain NPL-sgRNA; 2) Synthesize NPL-sgRNA forward and reverse oligonucleotide sequences, add BbsI enzyme cutting sites at both ends; form double-stranded oligonucleotides by gradient annealing; 3) Recover the Px459 plasmid vector after digestion with BbsI, and connect it with double-stranded NPL-gRNA by T4 ligase to form a recombinant gene knockout vector; single-clonal picking and sequencing of transformed competent strains to obtain the required NPL-gRNA/Cas9 recombination Knockout CRISPR vector, i.e. recombinant Px459 plasmid. 3. The method for constructing a sialidase gene knockout mouse model according to claim 2, wherein the NPL-sgRNA sequence is: GGTGTCCGCGGCGGAATCCG. 4. the construction method of a kind of sialidase gene knockout mouse model according to claim 2, is characterized in that, NPL-sgRNA forward and reverse oligonucleotide sequences are: Positive strand F: CACCGGGTGTCCGCGGCGGAATCCG; Anti-chain R: AAACCCGGATTCCGCCGCGGACACC. 5. The method for constructing the obtained sialidase gene knockout mouse as claimed in claim 1. 6. The application of the sialidase gene knockout mouse as claimed in claim 1 in the preparation of antitumor drugs.