CN105462970B - Pig specific friendly site Pifs501 and application thereof - Google Patents

Abstract

The invention relates to the field of genetic engineering, and particularly discloses a pig specific friendly site Pifs 501. The friendly site Pifs501 is positioned on the genome as follows: 44288819bp on chromosome 16 (NC-010458.3, based on pig genome sequence information version number Sscrofa10.2, 9 months 2011). The pig specific friendly site Pifs501 provided by the invention can be used for stable expression of exogenous genes, and solves the problems of unstable expression of the exogenous genes, unpredictability of position effect and the like in the transgenic process. The pig specificity friendly site Pifs501 provided by the invention ensures that the site-specific integration of exogenous genes on large livestock becomes reliable, and lays a foundation for the site-specific integration of large livestock. The invention can efficiently and specifically realize targeting by aiming at the targeting vector provided by the pig specific friendly site Pifs 501.

Description

Pig specific friendly site Pifs501 and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a pig specific friendly site Pifs 501.

Background

In the production process of transgenic animals, the key of the transgenic technology is whether the exogenous gene can realize efficient and stable expression in the transgenic animals. Exogenous genes are randomly integrated in the genome of a receptor animal, and are subjected to apparent modification to generate transgene silencing due to unstable expression quantity of position effect. In order to realize the continuous and stable expression of exogenous genes, site-directed transgenosis can solve various problems caused by position effects. In recent years, ZFN and TALEN technologies and a new CRISPR/Cas9 technology appear, so that a new tool is provided for the site-specific integration and gene knockout of genes, the transgenic efficiency is improved, and the success is achieved in multiple species. However, one of the keys to site-directed transgenesis is the selection of a good integration site that enables stable and consistent expression of different types of foreign gene expression cassettes without adversely affecting the animal cells or the individual animal. Currently there is less research on safe sites in large animals. It is therefore important to find such sites in pigs that are suitable for the integration of foreign genes.

The safe site in genome is one site in chromosome, and the transgene integrated in the site can be stably and reliably expressed in multiple tissues, and can adapt to the expression of different types of transgene expression cassettes, and has no adverse effect on endogenous gene structure and expression. However, the interaction between the foreign DNA and the host genomic sequence affects the reliability and safety of transgene integration. Although great progress has been made in transduction of a gene of interest, little research has been conducted on what sites in the genome of a recipient a gene is inserted to maximize safety and efficiency.

The CRISPR (clustered regularly interspersed short palindromic repeats)/Cas (CRISPR-associated) system is an immune system specific to prokaryotes and aims at exogenous genetic materials, and exogenous DNA (deoxyribonucleic acid) is cut and degraded through sequence-specific RNA mediation, and comprises bacteriophage and exogenous plasmids. The CRISPR/Cas system can be used as a gene editing system with site specificity, and has the biggest characteristics of simple operation, low cost and high effect. In 2013, scientists report that the CRISPR/Cas system is successfully applied to cells for the first time, and then the CRISPR/Cas system is rapidly applied to zebra fish, fruit flies, mice, rats and pigs. The CRISPR/Cas system generates double-stranded DNA break (DSB) at the target site, and the cell can be repaired by non-homologous end joining (NHEJ), resulting in frame shift mutation and loss of function of the gene. In addition, the system can also be used together with a homologous recombination vector and oligonucleotide to enable the target gene to be modified efficiently and accurately. In 2014, Scott JG et al realized gene replacement on zebrafish by using CRISPR/Cas-mediated homologous recombination. Hui Yang et al obtained reporter-bearing mice in one step using the same strategy. The CRISPR/Cas system is superior to a gene editing tool due to the great advantages, and is widely applied to the fields of gene function research, disease models, gene therapy and the like.

CRISPR/Cas9 mediated homologous recombination has achieved precise editing of genes on zebrafish, protozoa, mice, rats, but has not been reported on livestock large animals.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a pig specific friendly site Pifs501 and application thereof.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

in a first aspect, the invention provides a pig specific friendly site Pifs501, wherein the friendly site Pifs501 is located on a genome at the following positions: 44288819bp on chromosome 16 (NC-010458.3, based on pig genome sequence information version number Sscrofa10.2, 9 months 2011).

The friendly locus can be used for stable expression of exogenous genes, and solves the problems of unstable expression of the exogenous genes, unpredictability of position effect and the like in the transgenic process.

In a second aspect, the invention provides sgrnas that specifically target the friendly site Pifs 501.

And a DNA sequence for transcribing the sgRNA, wherein the nucleotide sequence is shown as SEQ ID No. 1.

In a third aspect, the invention provides a CRISPR/Cas9 targeting vector aiming at the friendly site Pifs501, wherein the targeting vector plays a role of a cutting vector.

Further, the construction method of the targeting vector comprises the following steps:

1) carrying out complementary pairing on the DNA sequences to form double-stranded DNA;

optionally, the DNA sequence is complementary paired with the nucleotide sequence shown in SEQ ID No.2 to form double-stranded DNA;

2) carrying out enzyme digestion on the px330 skeleton carrier with a restriction enzyme Bbs I overnight, recovering, and connecting with the double-stranded DNA obtained in the step 1).

The px330 backbone vector is a vector conventional in the art and is available from Addgene company.

Preferably, the present invention provides an optimal complementary pairing reaction procedure, specifically: denaturation at 94 ℃ for 5min, annealing at 35 ℃ for 10min, and preservation at 0-4 ℃. Alternatively, the reaction may be stored on ice.

In a fourth aspect, the invention provides a homologous recombination vector aiming at the friendly site, wherein the vector contains an exogenous target gene with homologous left arm and homologous right arm of Pifs501 site at two ends.

Further, the construction method of the homologous recombination vector comprises the following steps:

1) using pig cell genome DNA as a template, amplifying the homologous right arm of the Pifs501 site by using the primer sequences shown in SEQ ID No.3 and SEQ ID No.4, and amplifying the homologous left arm of the Pifs501 site by using the primer sequences shown in SEQ ID No.5 and SEQ ID No. 6;

2) the exogenous target gene is connected into the homologous recombination vector through NheI and NotI restriction enzymes.

In one embodiment of the invention, to verify the friendliness of the aforementioned site, the exogenous gene is the expression unit EGFP (reporter gene) from the pEGFP-N1 vector.

Furthermore, both ends of the exogenous target gene have loxP sites, one end is mutant loxP, the sequence is shown in SEQ ID No.7, and the other end is wild-type loxP.

In a fifth aspect, the invention provides a transgenic method, wherein the targeting vector and the homologous recombination vector are mixed according to the ratio of 1:1 of the material amount, and are co-transformed into porcine fibroblasts by an electric shock transfection or liposome transfection method.

The invention has the beneficial effects that:

the invention provides a pig specific friendly site Pifs501, wherein the friendly site can be used for stable expression of exogenous genes, does not cause gene silencing due to apparent modification, and solves the problems of instability of exogenous gene expression, unpredictability of position effect and the like in a transgenic process. The pig specificity friendly site Pifs501 provided by the invention ensures that the site-specific integration of exogenous genes on large livestock becomes reliable, and lays a foundation for the site-specific integration of large livestock. The invention can efficiently and specifically realize targeting by aiming at the targeting vector provided by the pig specific friendly site Pifs 501.

Drawings

FIG. 1 is a schematic diagram of the structure of the homologous recombination vector described in example 5 of the present invention.

FIG. 2 shows that the PCR method in example 6 of the present invention identifies a single cell, and a 7kb band is amplified when site-directed integration occurs, and a 3kb band is amplified when site-directed integration does not occur.

FIG. 3 is the expression of foreign genes at friendly sites in example 6 of the present invention.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Wherein: px330 vector was purchased from Addgene; t4DNA ligase and restriction enzyme are purchased from TaKaRa company; the primer synthesis and the sequence determination are completed by Shanghai Sheng worker and Shenzhen Huada; LongAmp Taq DNA polymerase, Q5DNA polymerase; trizol Reagent is available from Kangchi century Co; reverse transcriptase was purchased from Promega; SYBR Green from Roche; the plasmid endotoxin removing large extraction kit and the genome extraction kit are purchased from QIAGEN company; the conventional experimental operation steps of enzyme digestion, ligation, recovery, transformation, PCR amplification and the like are described in molecular cloning (third edition).

Example 1

Example 1 serves to illustrate the pig specific friendly site according to the invention.

The friendly site Pifs501 is positioned on the genome as follows: 44288819bp on chromosome 16 (NC-010458.3, based on pig genome sequence information version number Sscrofa10.2, 9 months 2011).

Example 2

Example 2 is intended to illustrate sgrnas specifically targeting the friendly site Pifs501 according to the present invention, which sgrnas are transcribed from the DNA sequence shown in SEQ ID No. 1.

Example 3

Example 3 serves to illustrate the CRISPR-Cas9 targeting vector for the friendly site Pifs501 described in the present invention.

The construction method of the CRISPR-Cas9 targeting vector comprises the following steps:

1. construction of targeting vectors

Complementary pairing is carried out on the sequence shown in SEQ ID No.1 and the sequence shown in SEQ ID No.2, and double-stranded DNA is constructed; the construction process is as follows: the oligonucleotides were stored at 94 ℃ for 5min, at 35 ℃ for 10min, and immediately on ice.

The px330 backbone vector was digested overnight with the restriction enzyme Bbs I, recovered and ligated to the annealed oligonucleotide for 3h at 16 ℃. And (4) converting and coating the plate by a conventional conversion method. After a single colony grows up, a plurality of colonies are picked for amplification culture and sequencing. The sequencing verifies correctly, and the successfully constructed CRISPR-Cas9 targeting vector is named as px 330-501.

Example 4

Example 4 is intended to illustrate the use of the targeting vector described in example 2, and specifically comprises the following steps:

1. amplification culture of positive single colony

The method comprises the following specific steps:

a. performing initial culture, picking positive single colony with a gun head, adding into a sterilization tube containing 5ml of LB culture medium (tryptone 10g, yeast extract 5g, NaCl 10g dissolved in 1L distilled water), and culturing at 37 deg.C and 220rpm for 8-12 h;

b. and (3) expanding culture, transferring the overnight culture solution into a sterilized triangular flask containing 100ml of LB culture medium according to the volume ratio of 1:500, and culturing at 37 ℃ and 220rpm for 12-16 h.

2. Large extraction of endotoxin from px330 plasmid

The px330 Plasmid was extracted according to the method provided on the Plasmid endotoxin removal large extraction Kit (EndoFree Plasmid Maxi Kit), and the extracted Plasmid was used for transfection of cells.

3. Cell transfection

Cell transfection was performed using Lonza Nucleofector. The specific process is as follows: a. digested and collected 6-well cellsMixing pig fibroblast (about 1 × 106) in one hole of the culture plate, 4 μ g of px330 plasmid and 100 μ l of nucleofector reagent, placing into an electric shock cup, and performing electric shock transfection by using a T-016 program; b. after the electric shock is finished, slowly adding 500 mu L of fibroblast culture medium (10% FBS + DMEM) preheated at 37 ℃ along the inner wall of the electric shock cup, and inoculating cells into one hole of the 6-hole cell culture plate; c. fibroblast culture medium (10% FBS + DMEM) without selection drug at 37.5 deg.C and 5% CO₂Culturing in an incubator.

Example 5

Example 5 is intended to illustrate the homologous recombination vector for the friendly site Pifs501 according to the invention.

The construction method of the homologous recombination vector is as follows:

1) using pig cell genome DNA as a template, amplifying the homologous right arm of the Pifs501 site by using the primer sequences shown in SEQ ID No.3 and SEQ ID No.4, and amplifying the homologous left arm of the Pifs501 site by using the primer sequences shown in SEQ ID No.5 and SEQ ID No. 6;

2) the expression unit EGFP from a pEGFP-N1 vector is used as a reporter gene (both ends of the expression unit EGFP are provided with loxP sequences, one end is mutant loxP, the sequence is shown as SEQ ID No.7, and the other end is wild-type loxP), and the expression unit EGFP is connected into a homologous recombination vector through NheI and NotI restriction enzyme to obtain the gene. The structure of the homologous recombination vector is schematically shown in FIG. 1.

Example 6

Example 6 is presented to illustrate the use of the targeting vector described in example 3 with the homologous recombination vector described in example 5 for transgenics.

1. Screening of Positive monoclonal cells

Digestion and Collection of one well of IBRS-2 cells or porcine fibroblasts (approximately 1X 10) in a 6 well cell culture plate⁶Respectively), mixing the constructed targeting vector and the homologous recombination vector according to the ratio of 1:1 of the amount of the substance, taking 4 mu g of the total mass, transfecting by the method in the embodiment 4, and then placing the mixture into CO₂Cultured in an incubator at 37.5 ℃. After 48h the confluency of the cells reached 80-90%, at which time 1 well of cells was divided into 8 10cm dishes on average. After 24h the cells were attached to the wall and the culture was allowed to proceedThe medium was changed to G418 (600. mu.g/mL) in fibroblast medium (10% FBS + DMEM) once every 3-4 days, and the medium was still G418 (600. mu.g/mL) in fibroblast medium. After 6-9 days of cell culture, cell clone point formation was observed. Finding a resistant cell cloning point under a microscope, marking by using a Marker pen, pouring out a culture medium, washing by using a PBS solution once, covering the resistant cell cloning point by using a cell cloning ring, adding 10-30 mu L of 0.25% trypsin digestion solution preheated at 37 ℃, digesting cells at 37.5 ℃ for about 2min, adding a cell culture medium to terminate digestion reaction, and inoculating the digested cells into a 48-hole cell culture plate for culture. When the cell confluence reaches 90%, digesting the cells, inoculating the cells into a 12-hole cell culture plate for continuous culture, continuously culturing the cells which are not digested in the original 48-hole cell culture plate for extracting cell genome DNA, continuously performing expansion culture on the cells to a 6-hole cell culture plate, and performing resistant cell cryopreservation according to a porcine embryo fibroblast cryopreservation method.

2. Identification of Positive cell monoclonals

Identification of the single clones of the cells picked:

using the extracted cell monoclonal genomic DNA as a template, PCR was performed using LongAmp Taq DNA polymerase to amplify a 7kb fragment, primers (5'-TGTGG ACTGT TGGCA AAG-3') and (5'-TTCTC GTGGA TTGGT GGA-3'), and PCR reaction using wild type cell genomic DNA as a template was used as a negative control. Amplification conditions: 94 ℃, 30 sec; 94 ℃, 30 sec; 58 ℃ for 30 sec; 7min at 65 ℃; 10min at 65 ℃; 35 cycles. After amplification was complete, the results were visualized by 0.8% agarose electrophoresis. The results are shown in FIG. 2. The upstream primer used in this step is located upstream of the homologous left arm, and the downstream primer is located downstream of the homologous right arm, so that a 7kb fragment can be amplified if homologous recombination occurs, and a 7kb fragment cannot be amplified if recombination does not occur.

In this example, the result of PCR identification across homology arms indicates that the exogenous gene is integrated at the target site.

For the cell clone with successful site-specific integration, we have adopted quantitative PCR method to analyze the exogenous gene expression, which shows that friendly site can support the high-efficiency expression of exogenous gene. As shown in fig. 3.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (8)

1. The sgRNA of the specific targeted pig friendly site Pifs501 is characterized in that a nucleotide sequence for transcribing a DNA sequence of the sgRNA is shown in SEQ ID No.1, and the position of the friendly site Pifs501 on a genome is as follows: 44288819bp on chromosome 16.

2. The CRISPR/Cas9 targeting vector for the pig specific friendly site Pifs501 is characterized in that the construction method comprises the following steps:

1) carrying out complementary pairing on the DNA sequence shown in SEQ ID No.1 to form double-stranded DNA;

2) carrying out enzyme digestion on the px330 skeleton carrier with a restriction enzyme Bbs I overnight, recovering, and connecting with the double-stranded DNA obtained in the step 1).

3. The method for constructing a targeting vector according to claim 2, comprising the steps of:

1) carrying out complementary pairing on the DNA sequence shown in SEQ ID No.1 to form double-stranded DNA;

2) carrying out enzyme digestion on the px330 skeleton carrier with a restriction enzyme Bbs I overnight, recovering, and connecting with the double-stranded DNA obtained in the step 1).

4. The construction method according to claim 3, wherein the reaction procedure of the complementary pairing is: denaturation at 94 ℃ for 5min, annealing at 35 ℃ for 10min, and preservation at 0-4 ℃.

5. The homologous recombination vector for friendly sites is characterized by comprising an exogenous target gene with Pifs501 site homologous left arm and homologous right arm at two ends, and the construction method comprises the following steps:

1) using pig cell genome DNA as a template, amplifying the homologous right arm of the Pifs501 site by using the primer sequences shown in SEQ ID No.3 and SEQ ID No.4, and amplifying the homologous left arm of the Pifs501 site by using the primer sequences shown in SEQ ID No.5 and SEQ ID No. 6;

2) the exogenous target gene is connected into the homologous recombination vector through NheI and NotI restriction enzymes.

6. The method for constructing the homologous recombination vector according to claim 5, comprising the steps of:

1) using pig cell genome DNA as a template, amplifying the homologous right arm of the Pifs501 site by using the primer sequences shown in SEQ ID No.3 and SEQ ID No.4, and amplifying the homologous left arm of the Pifs501 site by using the primer sequences shown in SEQ ID No.5 and SEQ ID No. 6;

2) the exogenous target gene is connected into the homologous recombination vector through NheI and NotI restriction enzymes.

7. The method of claim 6, wherein the foreign target gene has loxP sites at both ends, one end is mutant loxP site, the sequence is shown in SEQ ID No.7, and the other end is wild-type loxP site.

8. A transgenic method, characterized in that the targeting vector of claim 2 and the homologous recombination vector of claim 5 are mixed in a ratio of 1:1, and are co-transfected into pig fibroblasts by electroporation or lipofection.

Images