CN106701830B

CN106701830B - Pig embryo p66 knock-outshcMethod for gene

Info

Publication number: CN106701830B
Application number: CN201611115252.4A
Authority: CN
Inventors: 胡军和; 晏娇; 李甲梅
Original assignee: Hunan University of Humanities Science and Technology
Current assignee: Hunan University of Humanities Science and Technology
Priority date: 2016-12-07
Filing date: 2016-12-07
Publication date: 2020-01-03
Anticipated expiration: 2036-12-07
Also published as: CN106701830A

Abstract

The invention provides a method for knocking out p66shc in a main embryo by using a CRISPR-Cas9 gene editing method, which comprises the following specific operation steps: (1) designing an Oligo DNA sequence, (2) carrying out Oligo ligation, (3) transforming and amplifying the constructed plasmid, (4) verifying knockout efficiency of a plasmid transfected cell, (5) constructing a CRISPR-Cas9 system of a p66shc gene, (6) producing a pig p66shc gene knockout embryo, and (7) verifying a p66shc gene knockout result; by adopting the scheme, the ROS level in the in-vitro embryo culture process is effectively reduced, so that the in-vitro production efficiency of the pig embryo is improved by reducing the damage and the like in the in-vitro culture process, and finally, an effective in-vitro production system of the pig embryo is constructed.

Description

Pig embryo p66 knock-outshcMethod for gene

Technical Field

The invention mainly relates to the field of embryo engineering technology and genetic engineering, in particular to a method for constructing knock-out p66 by using a CRISPR/Cas9 gene editing method^shcA method for gene pig embryo.

Background

Early embryo development is largely related to Reactive Oxygen Species (ROS) levels in the culture environment. The existing research results show that the existence of the hydrogen peroxide can induce the mouse fertilized eggs to generate biological events such as periodic arrest, apoptosis, necrosis and the like in the in-vitro culture process^[13]. Mild oxidative damage initially leads to depolarization of the inner mitochondrial membrane, altered mitochondrial matrix and intracellular mitochondrial distribution; research finds that the product uses H₂O₂After 48h of treatment, the fertilized eggs are shriveled, and after 72h, the development is stopped, and DNA fragments appear, which indicates that mitochondria are damaged by oxidation in the early stage of embryonic development, so that mitochondria dysfunction can be caused, and the main reasons of cell cycle arrest and apoptosis can be caused^[13]. Research shows that embryo p66^shc(66-kilodahon iso of Shc gene products) expression has a close relationship with ROS level, and it was found that p66 was found in the early embryonic development-retardant middle embryo of cattle^shcmRNA levels significantly higher than normal embryos^[10]. Approximately 14% of bovine in vitro embryo production blocks at the 2-4 cell stage of division, and a large number of ROS are found in these blocked embryos, mainly as H₂O₂In the form that the production and amount of these ROS increases as the embryo develops in vitro; the ROS levels in vitro-developing embryos are much higher relative to in vivo embryos, and the produced ROS can adversely affect the embryos, cause DNA chain breakage, increase the oxidation level of proteins and the like^[17,18]. Studies have also shown that the level of ROS produced can cause embryogenesis to die, affecting the efficiency of embryonic in vitro development, and that p66 has been shown^shcCapable of recognizing DNA damage caused by ROS^[1]. For this purpose, the p66 is lowered inside the embryo^shcThe expression of (3) can reduce the generation of ROS in the embryo, thereby improving the in vitro development efficiency of the pig embryo.

It is well known that the CRISPR-Cas system (clustered, short palindromic repeats-associated protein systems) is an adaptive mechanism that has evolved from bacteria and archaea to protect against viral and plasmid invasion. The highly efficient genome editing functions of the CRISPR-Ca system have been applied to a variety of organisms including zebrafish, mouse, rat, caenorhabditis elegans, plants and bacteria. Studies by various research groups have shown that CRISPR-Cas system-mediated genome targeting experiments show similar or even higher efficiency in cells or zebrafish compared to Zinc Finger Nucleases (ZFNs) and transcription-activator-like effector nucleases (TALENs). The RNA-DNA recognition mechanism of CRISPR-Cas9 system provides a simple and powerful tool for selective genome editing. In practical applications, the CRISPR/Cas system usually combines two genes encoding a trancrrna and a crRNA into a single-stranded guide rna (sgRNA), i.e., the currently used CRISPR/Cas system mainly consists of Cas9 protein and sgRNA. One of the most important advantages of this system is that the Cas9 protein can simultaneously modify multiple genomic targets under the guidance of multiple different sgrnas (small guide rnas).

Based on the thought, the p66 is knocked out by using a CRISPR/Cas9 gene editing method in the early embryo stage (prokaryotic injection method)^shcThe gene reduces the ROS expression of the embryo, and subsequent research work is carried out by screening positive embryos, so that a high-efficiency in-vitro production technology system of the pig embryos is finally established.

Disclosure of Invention

The invention aims to construct a knock-out p66^shcThe gene pig embryo production system reduces the ROS level in the in vitro embryo culture process, thereby reducing the damage and the like in the in vitro culture processHigh efficiency of in vitro production of pig embryo, and finally constructing an effective in vitro production system of pig embryo.

In order to realize the purpose of the invention, the invention firstly provides a method for knocking out p66 in a main embryo by using a CRISPR-Cas9 gene editing method^shcThe method comprises the following specific operation steps:

(1) design of Oligo DNA sequences

At p66^shcA pair of oligo DNAs of about 20bp is designed in the expression DNA region of the gene, and can be designed by an on-line tool. The Oligo synthesis of 1-3 pairs is preferentially selected according to the score. In addition, a primer is required to be designed on the upstream and the downstream of the site for detecting positive cloning in subsequent PCR or sequencing, the primer can amplify a DNA fragment of about 300bp, the upstream primer is about 100bp away from the mutation site, and the downstream primer is about 200bp away from the mutation site. The designed sequence was sent to a trusted company for synthesis, and the purification grade was PAGE, ready for the following experiments.

(2) Ligation of Oligo

The synthesized 2 single-stranded Oligo DNAs were diluted to 1. mu.g/. mu.l, annealed to form double-stranded DNAs, and ligated to a vector. pGLKO (purchased from Genloci) is a linear vector, does not require an enzyme digestion treatment, and can be used directly for T4 DNA ligase ligation reaction.

(3) Constructed plasmid transformation and amplification

Transforming the product into Escherichia coli competent cells according to conventional molecular cloning technique, screening positive clones, selecting positive clones, amplifying and culturing, and extracting large amount of plasmids to obtain constructed strain containing knock-out p66^shcAnd (3) an expression plasmid of the CRISPR-Cas9 system of the gene is stored for later use.

(4) Validation of knockout efficiency in plasmid transfected cells

The 293T cells are thawed and cultured firstly, and transfection operation is carried out after 2 passages of growth and culture. On the premise of cotransfection, diluting to the plasmid concentration of 1 mug/mug, inoculating cells to a pore plate or a culture dish for standby slow virus production 48 hours before transfection, and when transfection is carried out, the cell confluency is about 70% -80% and is in the optimal infection state, and the activity is more than or equal to 95%. The supernatant is harvested after 24h of one-time harvest by taking the transfection time as the starting pointAnd stored at 4 deg.C, or-84 deg.C for long term storage. Inoculating appropriate amount of target cells, adding the collected virus particle solution containing plasmid, and mixing. After the target cells are subjected to limiting dilution, the target cells are distributed into a 96-well plate for monoclonal culture, and after the cells grow well, about 1000 cells are taken to extract genomes. Then, positive clones were preliminarily screened using a T7 endonuclease, and the screened positive clones were subjected to sequencing analysis and base deletion results were subjected to alignment analysis, whereby the p66 could be verified^shcEffectiveness of the gene CRISPR-Cas9 system.

(5)p66^shcCRISPR-Cas9 system construction of gene

The primers of Cas9 on the plasmid and a Kit mMESSAGENMAXIANHINE Ultra Kit (Ambion) are designed to amplify the obtained Cas9 mRNA, and meanwhile, the sequences of gRNA are amplified by the designed primers by using a Kit MEGAshortscript (Ambion). After obtaining PCR products, the PCR products were electrophoresed on 1.5% gels, respectively, and the sizes of the obtained fragments were confirmed. To obtain p66^shcThe CRISPR-Cas9 system mRNA of the gene is stored for later use.

(6) Pig p66^shcGene knock-out embryo production

The constructed porcine embryos were injected cytosolically 17h after fertilization with p66 as described above^shcCRISPR-Cas9 system mRNA of genes, at a concentration of 10ng/ul, was injected in a total amount of 3ul per embryo. After culturing, the subsequent system is used to test the knockout effect of the gene and evaluate the knockout efficiency.

(7)p66^shcGene knockout result verification

P66 after PCR cloning of blastocysts^shcThe DNA sequence of the gene (generally the product is better at 0.5-1K), the primer design of the gene is careful that the mutation site of the obtained product is not well positioned in the middle, and the product is confirmed to be knocked out or not by gel electrophoresis analysis after being cut by T7 endonuclease. Meanwhile, the PCR product can be used for sequencing and then contrast analysis to draw a conclusion.

Drawings

FIG. 1 shows the screening of three pairs of p66^shcOligo primer of CRISPR-Cas9 system of genes.

FIG. 2 is a diagram of the vector structure involved in the assay for linking gRNAs and expressing mRNA synthesis template sequences for embryo injection.

FIG. 3 shows the construction p66 for verifying it^shcEnzymatic cleavage schematic of knockout efficiency of CRISPR-Cas9 system of genes.

Detailed Description

(1) Design of Oligo DNA sequences

First, total DNA was extracted from porcine aborted fetal tissues by designing p66^shcPCR cloning of upstream and reverse downstream primers of Gene p66^shcThe full length of the gene is then subjected to DNA sequencing, the sequence is preserved, and the promoter region is analyzed for future use. Then, at p66^shcA pair of oligo DNAs of about 20bp is designed in the expression DNA region of the gene, and can be designed by the following online tool: CRISPR Design (http:// CRISPR. mit. edu /) from massachusetts institute of technology or E-Crisp (www.e-Crisp. org/E-Crisp/designrispr. html) from german center for cancer research. The Oligo synthesis of 1-3 pairs is preferentially selected according to the score. By on-line design, the following better 3 pairs of Oligo were selected:

(1).TCAATAAGCCCACGCGGGGCTGG

(2).CCGGGGTTTCCTACTTGGTTCGG

(3).GCGAGGAGTGGACCCGCCACGGG

thus, the following 3 pairs of primers were used according to the requirements for plasmid ligation

(1)p66^shc-F:5’…accgGTCAATAAGCCCACGCGGGGC…3’

p66^shc-R:5’…aaacGCCCCCGCGTGGGCTTATTGAC…3’

(2)p66^shc-F:5’…accgGCCGGGGTTTCCTACTTGGTT…3’

p66^shc-R:5’…aaacGAACCAAGTGGAAACCCCGGC…3’

(3)p66^shc-F:5’…accgGCGAGGAGTGGACCCGCCACG…3’

p66^shc-R:5’…aaacGCGTGGCGGGTCCACTCCTCGC…3’

The designed sequence was sent to a trusted company for synthesis, and the purification grade was PAGE, ready for the following experiments.

(2) T4 DNA Ligase ligation

The synthesized 2 single-stranded Oligo DNAs were diluted to 1. mu.g/. mu.l, annealed to form double-stranded DNAs, and ligated to a vector. The pGLKO linear vector can be directly used for T4 DNA ligase ligation reaction without enzyme digestion treatment, and an annealing reaction system is as follows:

the system is subjected to instantaneous centrifugation, placed in a PCR instrument and incubated at 95 ℃ for 3min, and naturally cooled for 20min after incubation. Taking 1.75. mu.l of hybridized double-stranded DNA to perform T4 ligase ligation, wherein the reaction system is as follows:

(3) constructed plasmid transformation and amplification

The product is transformed into escherichia coli competent cells according to a conventional molecular cloning technology, and the specific operation process is as follows: a single colony of DH5 alpha is picked on an LB plate and inoculated in 3.0mL of LB liquid medium, and is subjected to shaking culture at 200rpm overnight; inoculating the strain into a new LB culture medium according to the volume of 2% the next day, and performing shake culture at 200rpm for 3-4 h until OD600 is 0.3-0.5; transferring the bacteria into a 1.5mL sterilized centrifuge tube, wherein each tube contains 1.0mL of bacteria, and carrying out ice bath for 30 min; centrifuging at 12000rpm at 4 deg.C for 30 s; completely discarding the supernatant, resuspending the bacteria with 1.0mL of 0.1mol/L ice-cold CaCl2, and ice-cooling for 10 min; centrifuging at 12000rpm at 4 deg.C for 30 s; the supernatant was discarded completely, and the bacteria were resuspended in 100. mu.L of 0.1mol/L ice-cold CaCl2 and kept at 4 ℃ in a refrigerator for 24 h. Adding 1.0 μ L (proper amount) of plasmid into 100 μ L of freshly prepared competent cells of Escherichia coli, mixing gently, and ice-cooling for 30 min; then putting the mixture into a water bath with the temperature of 42 ℃ for heat shock for 90s, and quickly cooling the mixture in ice for 1-2 min; adding preheated 800 μ L LB culture medium into each tube, and shake culturing at 37 deg.C and 200rpm for 45 min; and (3) taking 100 mu L of bacterial liquid, then coating the bacterial liquid on a corresponding resistant plate, and culturing for 16-24 h at 37 ℃.

Screening positive clones, selecting the positive clones, amplifying and culturing, and extracting a large amount of plasmids, wherein the specific operation steps are as follows: single colonies on the coated resistant plates were transformed by picking the ligation product of the foreign gene and the vector, inoculated in 3.0mL of LB medium containing 50. mu.g/mL of ampicillin, and cultured with shaking overnight at 200 rpm. Taking 1.5mL of bacterial liquid, centrifuging at 12000rpm for 30s, and precipitating thalli; after the supernatant was completely discarded, 300. mu.L of 4 ℃ precooled solution I (50mmol/L glucose; 25mmol/L Tris-Cl, pH 8.0; 10mmol/L EDTA) was added to resuspend the bacteria; adding 300 μ L (0.2mol/L NaOH; 1% SDS) of the newly prepared solution II, rapidly inverting the centrifuge tube for 5 times, and placing the centrifuge tube on ice; adding 225 μ L of ice-precooled solution III (5mol/L potassium acetate 60mL, glacial acetic acid 11.5mL, water 28.5mL), covering the tube opening tightly, inverting the tube, and then turning the tube upside down to denature the protein into white lumps, and placing the white lumps on ice for 3-5 min; centrifuging at 12000rpm at 4 deg.C for 5min, and transferring the supernatant to a new centrifuge tube; adding phenol and chloroform with the same volume, reversing, mixing uniformly, placing at room temperature for 5min, and centrifuging at 12000rpm for 5 min; taking the supernatant to a new centrifuge tube, adding isopropanol with the same volume, reversing and mixing uniformly, and standing at room temperature for 10 min; centrifuging at 12000rpm for 0min, removing supernatant, washing precipitate with 70% ethanol, drying, and dissolving with 30 μ L TE (pH8.0) or double distilled water; mu.L of RNase A (10mg/mL) was added thereto and allowed to act at 37 ℃ for 30 min.

The constructed inclusion knock-out p66 was obtained^shcAnd (3) an expression plasmid of the CRISPR-Cas9 system of the gene is stored for later use.

(4) Validation of knockout efficiency in plasmid transfected cells

The 293T cells are thawed and cultured firstly, and transfection operation is carried out after 2 passages of growth and culture. On the premise of cotransfection, diluting to the plasmid concentration of 1 mug/mug, inoculating cells to a pore plate or a culture dish for standby slow virus production 48 hours before transfection, and when transfection is carried out, the cell confluency is about 70% -80% and is in the optimal infection state, and the activity is more than or equal to 95%. The specific operation process is as follows:

(1) adding 800ul of 0.25% pancreatin for digestion for about 30s, observing cell morphology at any time, adding a complete culture medium with 2 times of pancreatin volume to stop digestion after digestion is finished, and uniformly blowing and beating;

(2) centrifuging at 5 deg.C and 900rpm for 5min, and removing supernatant;

(3) adding 1ml PBS, after resuspending, centrifuging for 5min at 25 ℃, 900rpm, removing supernatant, and repeating the step for 1-2 times;

(4) add 200ul serum-free medium, resuspend cells, add 5. mu.l each of pGLKO linear vector (Genloci Co.) and Packaging Mix (Genloci Co.);

(5) in the centrifugation process, preparing an electric transfer device, turning on a power supply, sucking 200ul of cell suspension into an electrode tube by a liquid transfer gun (ensuring no bubbling), and carrying out electric transfer according to the electric transfer conditions of 1200V, 20ms and 2 pulses;

(6) after the electrotransfer is finished, putting the mixture into a prepared pore plate or a culture dish, adding a proper amount of DMEM medium containing 2% -10% of serum concentration, and culturing the mixture in a cell culture box.

The supernatant was harvested after 24h of one harvest time with the transfection time as the starting point and stored at 4 ℃ or-84 ℃ for long term storage. Inoculating appropriate amount of target cells, adding the collected virus particle solution containing plasmid, and mixing. After the target cells are subjected to limiting dilution, the target cells are distributed into a 96-well plate for monoclonal culture, and after the cells grow well, about 1000 cells are taken to extract genomes. Then, positive clones were preliminarily screened using a T7 endonuclease, and the screened positive clones were subjected to sequencing analysis and base deletion results were subjected to alignment analysis, whereby the p66 could be verified^shcEffectiveness of the gene CRISPR-Cas9 system.

(5)p66^shcCRISPR-Cas9 system construction of gene

(6) Pig zygote embryo injection

Subjecting the constructed pig embryo toCytosolic injection of p66 described above 17h after sperm^shcCRISPR-Cas9 system mRNA of genes, at a concentration of 10ng/ul, was injected in a total amount of 3ul per embryo. And comparative injections were performed according to the CRISPR-Cas9 system, embryome group, buffered embryome group and non-embryome group. After injection, culturing for 4-6 hr, observing survival condition of embryo, selecting good quality embryo for subsequent culture, and observing after 6 days.

(7) Comparison of embryo development efficiency

Injection of p66^shcThe CRISPR-Cas9 system embryome group, the group of injected buffered embryos, and the group of non-injected embryos (control group) of genes were subsequently cultured in vitro. Washing the embryo culture solution for 2-3 times, transferring into 25 μ L microdroplets, covering with mineral oil, and culturing 1-2 embryos per drop at 38.5 deg.C with saturated humidity and 5% CO 2. And recording the number of cleavage after 44-48 h, recording the number of morulae and blastula on

days

6 and 7 respectively, and observing the cleavage rate, the blastula rate and other indexes of different groups.

(8)p66^shcGene knockout result verification

Selecting embryos which develop to blastocyst, and detecting p66 by RT-PCR through sucking 5-10 trophoblast cells^shcWhether a gene is expressed or not was calculated, and the efficiency of its knock-out. P66 after PCR cloning of blastocysts^shcThe DNA sequence of the gene (generally the product is better at 0.5-1K), the primer design of the gene is careful that the mutation site of the obtained product is not well positioned in the middle, and the product is confirmed to be knocked out or not by gel electrophoresis analysis after being cut by T7 endonuclease. Meanwhile, the PCR product can be used for sequencing and then contrast analysis to draw a conclusion.

The following table is p66^shcOligo primer design for CRISPR-Cas9 system of genes

。

Claims

1. Pig embryo p66 knock-out^shcA method of genomics, characterized by: the method comprises the following operational steps of,

(1) design of Oligo DNA sequences

Designing a pair of oligo DNAs (deoxyribonucleic acids) of about 20bp in an expression DNA region of a p66shc gene, wherein the oligo DNAs can be designed through an online tool; synthesizing Oligo in 1-3 pairs according to the scores; in addition, a primer is required to be designed on the upstream and the downstream of the site respectively for detecting positive cloning in subsequent PCR or sequencing, the primer can amplify a DNA fragment of about 300bp, the upstream primer is about 100bp away from the mutation site, and the downstream primer is about 200bp away from the mutation site; sending the designed sequence to a reliable company for synthesis, wherein the purification level is PAGE, and preparing for the following experiments;

(2) ligation of Oligo

Diluting the synthesized 2 single-stranded Oligo DNAs to 1 mug/mul, annealing to form double-stranded DNAs, and connecting the double-stranded DNAs with a carrier; pGLKO is a linear vector, does not need enzyme digestion treatment and can be directly used for T4 DNA ligase ligation reaction;

(3) constructed plasmid transformation and amplification

Transforming the product into an escherichia coli competent cell according to a conventional molecular cloning technical method, screening positive clones, picking out positive clones, performing amplification culture, extracting a large amount of plasmids to obtain a constructed expression plasmid containing a p66shc gene-knocked-out CRISPR-Cas9 system, and storing for later use;

(4) validation of knockout efficiency in plasmid transfected cells

Firstly, thawing and culturing 293T cells, and performing transfection operation after growth and culture passage for 2 times; on the premise of cotransfection, diluting until the concentration of plasmid is 1 mug/mug, inoculating cells to a pore plate or a culture dish for standby production of slow virus 48 hours before transfection, wherein the confluence degree of the cells is 70-80% in the process of transfection, the cells are in the optimal infection state, and the activity is more than or equal to 95%; taking the transfection time as a starting point, harvesting the supernatant after 24h of one-time harvesting time, and storing at 4 ℃, if the supernatant is stored for a long time, the supernatant is stored at-84 ℃; inoculating a proper amount of target cells, adding the collected virus particle solution containing the plasmids, and uniformly mixing; after limiting dilution, dividing the target cells into 96-well plates for monoclonal culture, and after the cells grow well, taking about 1000 cells and extracting a genome; then, primarily screening positive clones by using a T7 endonuclease, performing sequencing analysis on the screened positive clones, and performing comparison analysis on a base deletion result, so that the effectiveness of the p66shc gene CRISPR-Cas9 system can be verified;

(5) CRISPR-Cas9 system construction of p66shc gene

Designing a primer of Cas9 on the plasmid, amplifying the obtained Cas9 mRNA by using a Kit mMESSAGE mMACHINE Ultra Kit, and amplifying a gRNA sequence by using the designed primer and the Kit MEGA shortscript; after PCR products are obtained, respectively carrying out electrophoresis by using 1.5% gel, and identifying the sizes of the obtained fragments; after CRISPR-Cas9 system mRNA of the p66shc gene is obtained, storing for later use;

(6) production of pig p66shc gene knockout embryo

Carrying out cytoplasm injection on the constructed porcine embryos 17h after fertilization on CRISPR-Cas9 system mRNA of the p66shc gene, wherein the concentration is 10ng/ul, and the total amount of each embryo is 3 ul; after culturing, the subsequent system is used for checking the knockout effect of the gene and evaluating the knockout efficiency;

(7) verification of p66shc gene knockout result

The DNA sequence of the p66shc gene after the blastocyst is cloned by PCR, the primer design of the DNA sequence is good, the mutation site of the obtained product is not well positioned in the middle, and after the T7 endonuclease is used for enzyme digestion, gel electrophoresis analysis is carried out to confirm whether the DNA sequence is knocked out or not; meanwhile, the PCR product can be used for sequencing and then contrast analysis to draw a conclusion.