CN111041047A - Construction method of mouse model for conditionally site-specific double overexpression HPV E6/E7 gene - Google Patents
Construction method of mouse model for conditionally site-specific double overexpression HPV E6/E7 gene Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- C12N15/90—Stable introduction of foreign DNA into chromosome
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention provides a construction method of a mouse model of conditional site-specific double overexpression HPV E6/E7 gene, which comprises the following steps: 1. respectively designing and obtaining sgRNAs, (2) respectively preparing a Cas9/sgRNA mixture; (3) constructing a targeting vector, (4) carrying out microinjection and obtaining with F0 generation mice, wherein the targeting vector comprises F0 generation mice of a ROSA26 site fixed point knock-in HPV E6 and F0 generation mice of an H11 site fixed point knock-in HPVE 7; (5) f1 mouse; (6) obtaining F2 generation positive homozygote mice; 2. obtaining a conditional site-directed HPV E6/E7 double knock-in model mouse: two F2 generation positive homozygous mice were crossed to obtain a conditional site-directed HPV E6/E7 double knock-in model mouse. The method provided by the invention can obtain an animal model which can be stably inherited and accurately regulated.
Description
Technical Field
The invention relates to a construction method of a mouse model of conditional site-specific double overexpression HPV E6/E7 gene, belonging to the technical field of biology.
Background
Cervical cancer is one of the most common malignant tumors of women, the incidence rate of the cervical cancer is second to breast cancer in gynecological malignant tumors, the incidence process is complex, the incidence rate is high, the incidence population tends to be younger, and the life health of women is seriously threatened. Its development is a complex, multi-step process, and is the result of long-term synergy of multiple carcinogens, including Human Papillomavirus (HPV) infection, genetic variation, immune escape, and the like. A large number of epidemiological and molecular biological research results prove that HPV and epithelial malignant tumor of genital tract are closely related to precancerous lesion and are the main etiological factors of cervical cancer. HPV has high tissue specificity and strong tropism to squamous epithelium. More than 200 subtypes of HPV have been identified and classified into high-risk type HPV (hrHPV) and low-risk type HPV (lrHPV) according to their oncogenicity. The gene structures of all subtypes are approximately similar and can be divided into 3 parts: early (E) coding region, late (L) coding region and long regulatory region (LCR). Among them, hrHPV E6 and E7 play a key role in the HPV carcinogenesis process, the encoded protein products are important factors for causing cervical epithelial canceration, and the two proteins influence the cell growth cycle and DNA repair by combining with intracellular oncostatin P53, pRb and the like, so that epithelial cells are promoted to exit the differentiation program and continuously proliferate. The method has important medical application value for carrying out deep research on the aspects of the mechanism of the generation and development process of the cervical cancer, the diagnosis and treatment of the cervical cancer, the research and the development of vaccines and the like of E6/E7.
At present, the bottleneck still exists in the research on how to comprehensively evaluate the cancer promotion mechanism of hrHPV E6/E7, especially the mechanism in the processes of inducing normal cell malignant transformation and tumor microenvironment immune regulation. In order to address the above scientific issues, the development of in vivo models is essential, and an organic whole of the in vivo environment is required for studying the interaction of molecules with lesions. The function of the in vivo hrHPV E6/E7 protein can be evaluated by the characteristics of relevant animal models, and the relationship between the hrHPV and female cervical dysplasia, cervical cancer and the like is discussed. At present, relevant researches are carried out by mostly utilizing nude mice and traditional random transgenic mice.
(1) Nude mice: domestic researchers induce the expression of E6/E7 in the uterus of a tested mouse by constructing an E6/E7 adenovirus vector with a keratin (K14) promoter, injecting recombinant virus Ad-K14-E6/E7 into a tail vein of a nude mouse in a mode of combining intraperitoneal injection of estrogen and the like.
(2) Transgenic mice: the transgenic animal refers to an animal in which an exogenous gene transferred by an experimental method is integrated in a genome, expressed and transmitted to a descendant, breaks the species limit, and provides possibility for researching genes from different sources. For example, researchers have constructed vectors carrying a skin tissue specific promoter PINVThe HPV16E 6/E7 eukaryotic expression vector of (Humaninvolucrinpromoter) is subjected to microinjection after linearization to obtain pINV-E6/E7 transgenic founder mice, and the proportion of positive offspring accords with Mendel's law of inheritance.
In the above model, the nude mouse lacks a robust immune system, and the model is not suitable for studying the role of HPV E6/E7 in mediating tumor microenvironment immune regulation, immune escape and the like. However, the conventional random transgenic mice have been limited in many fields. The efficiency of the transgene and the uncertainty of the integration site are the main reasons. The mice are generally prepared by injecting germ cells into pronuclei by plasmids, integration of the mice is random, some mice occur in a single site, and the integration of the mice is also integrated on a plurality of sites of different chromosomes under the condition of random, genome rearrangement and translocation deletion can be caused by the randomness of the integration of the; in addition, the copy number of integration is not fixed, and multi-copy tandem integration may cause low expression rate or even no expression of exogenous genes, which is not favorable for controlling the expression level and function research of genes. Therefore, the first established mouse (positive genotype identification) is obtained and needs further screening.
Disclosure of Invention
The invention aims to provide a method for constructing a mouse model of conditional site-specific dual over-expression HPV E6/E7 gene, so as to solve the problems.
The invention adopts the following technical scheme:
a method for constructing a mouse model of conditional site-specific dual overexpression HPV E6/E7 gene is characterized by comprising the following steps:
1. obtaining mice with conditional site-directed tapping of the ROSA26 site HPV E6 and H11 site-conditional site-directed tapping HPVE 7:
(1) designing and obtaining sgRNA at mouse ROSA26 site and H11 site respectively according to the sequences of HPV E6 and E7 genes, and evaluating the off-target effect of candidate sequences;
(2) respectively incubating sgRNAs corresponding to the two sequences with Cas9 protein to respectively prepare Cas9/sgRNA mixtures;
(3) constructing a targeting vector, and constructing homologous recombination vectors carrying conditional overexpression E6 or E7 sequences respectively by utilizing In-Fusion cloning technology, wherein a recombination system used In the construction process is a Cre-LoxP induced expression system, and the vectors comprise 'start-loxP-Stop-loxP-Kozak-HPV E6 or E7-screening marker-polyA' gene segments;
(4) microinjection and F0 generation mice, respectively, and the targeting vector and the Cas9/sgRNA mixture are injected into fertilized eggs of the mice together; then sending the fertilized eggs after microinjection back to the oviduct of a surrogate mouse, and after the mouse is born, screening out a target mouse through gene identification, namely an F0 generation mouse, including an F0 generation mouse of which the site of ROSA26 is knocked into HPV E6 at a fixed point and an F0 generation mouse of which the site of H11 is knocked into HPV E7 at a fixed point;
(5) obtaining mice of F1 generation, breeding the sexually mature positive F0 generation mice with wild mice to generate F1 generation, and obtaining F1 generation positive heterozygote mice after PCR and Southern verification, wherein the F1 generation positive heterozygote mice comprise F1 generation mice of ROSA26 site fixed point knock-in HPV E6 and F1 generation mice of H11 site fixed point knock-in HPV E7;
(6) selfing the F1 generation positive heterozygote mouse with the site of ROSA26 knocked-in HPV E6 and the F1 generation positive heterozygote mouse with the site of H11 knocked-in HPV E7, and screening to obtain an F2 generation positive homozygote mouse;
2. obtaining a conditional site-directed HPV E6/E7 double knock-in model mouse:
the mouse with the ROSA26 site-specific knock-in HPV E6F 2 generation positive homozygote is hybridized with the mouse with the H11 site-specific knock-in HPV E7F2 generation positive homozygote to obtain a conditional site-specific HPV E6/E7 double knock-in model mouse.
Further, the method for constructing the mouse model for conditionally overexpressing HPV E6 gene at the ROSA26 site also has the following characteristics:
the sequences of sgrnas conditionally overexpressing HPV16E6 are:
CUCCAGUCUUUCUAGAAGAUGGG,
the sequences of sgrnas conditionally overexpressing HPV16E7 are:
GAACACUAGUGCACUUAUCCUGG。
further, the method for constructing the mouse model for conditionally overexpressing HPV E6 gene at the ROSA26 site of the invention also has the following characteristics:
mouse genotype identification: by adopting PCR detection or sequencing verification,
extracting genome DNA of an E6 mouse rat tail, and performing PCR identification after the target of the repair donor is in the target, wherein primers ROSA26-seqF1/ROSA26-seqR1 are respectively positioned outside a 5 'homology arm and in a repair donor fragment, and if a PCR product is generated by amplification of the pair of primers, the target donor is effectively inserted in the 5' genome of the mouse; ROSA26-seqF2/ROSA26-seqR2 are respectively positioned in the fragment of the repair donor and outside the 3 'homology arm, if the pair of primers are amplified to generate a PCR product, the target donor is effectively inserted into the 3' of the mouse genome;
e7 mouse rat tail genomic DNA was extracted and subjected to PCR identification after targeting with repair donor. As shown in FIG. 19, primers H11-seqF1/H11-seqR1 are respectively positioned outside the 5 'homology arm and in the repair donor fragment, and PCR products are generated by the amplification of the primers, so that the target donor is effectively inserted in the 5' of the mouse genome; H11-seqF2/H11-seqR2 are respectively positioned in the fragment of the repair donor and outside the 3 'homology arm, and if the pair of primers is amplified to generate a PCR product, the target donor is effectively inserted in the 3' of the mouse genome.
Further, the construction method of the mouse model for conditionally overexpressing HPV E6 gene at the ROSA26 site of the invention also has the characteristics that the primer sequences adopted by PCR detection or sequencing verification are as follows:
e6 mice:
e7 mice:
primer name | Primer sequences |
F1 | 5’-GTACATCCACAGCATCTTCCAAG-3’ |
R1 | 5’-AGATGTACTGCCAAGTAGGAAAGTC-3’ |
F2 | 5’-GCATCTGACTTCTGGCTAATAAAG-3’ |
R2 | 5’-GCCTTGACCTAAGAGATGATGCGAC-3’ |
F3 | 5’-CTCTACTGGAGGAGGACAAACTG-3 |
R3 | 5’-GTCTTCCACCTTTCTTCAGTTAGC-3’ |
Further, the method for constructing a mouse model for conditionally overexpressing HPV E6 gene at the ROSA26 site of the invention is characterized in that if the purity of a DNA sample is low or the PCR reaction time is desired to be shortened, alternative PCR primers are adopted, and a PCR product with a shorter fragment can be obtained, wherein the sequences of the alternative PCR primers are respectively as follows:
e6 mice:
e7 mice:
further, the construction method of the mouse model for conditionally overexpressing HPV E6 gene at the ROSA26 site also has the characteristics that an alternative PCR reaction system:
reaction assembly | Volume of |
MousetailgenomicDNA | 1μL |
Forwardprimer(10μM) | 1μL |
Reverseprimer(10μM) | 1μL |
PremixTaqPolymerase | 12.5μL |
ddH2O | 9.5μL |
Total | 25μL |
Further, the method for constructing the mouse model for conditionally overexpressing HPV E6 gene at the ROSA26 site also has the characteristics that the PCR reaction conditions are selected as follows:
further, the method for constructing the mouse model for conditionally overexpressing HPV E6 gene at the ROSA26 site of the invention also has the following characteristics:
mouse genotype identification: and (3) identifying by using southern blot:
taking the double-arm homologous recombination positive mouse (No. 2-4) verified by PCR and sequencing, extracting rat tail DNA, carrying out enzyme digestion by restriction enzyme BamHI or BstEII, simultaneously selecting a DNA probe for Southern blot analysis,
e6 mouse 5' probe primer sequence:
e6 mouse 3' probe primer sequence:
e7 mouse KI probe primer sequence:
further, the method for constructing the mouse model for conditionally overexpressing HPV E6 gene at the ROSA26 site of the invention also has the following characteristics:
crossing the fixed point HPV E6 single knock-in mouse, the fixed point HPV E7 single knock-in mouse or the fixed point HPV E6/E7 double knock-in model mouse with Cre mouse with tissue specific promoter or infecting with Cre virus.
Advantageous effects of the invention
Aiming at the problems in the prior art, the invention provides a method for establishing an HPV E6/E7 conditional site-specific double-knock-in mouse model by applying a CRISPR-Cas9 genome site-specific editing technology so as to obtain an animal model which can be stably inherited and accurately regulated. Compared with the prior art, the invention mainly solves the following technical problems:
the problem that a nude mouse lacks a healthy immune system and is not suitable for researching the effect of HPV E6/E7 in the process of mediating tumor microenvironment immune regulation, immune escape and the like in the prior art is solved, and the animal model provided by the invention has a healthy immune system and is suitable for all researches on the interaction of HPV E6/E7 and the immune system;
the problem that the gene efficiency and the integration site uncertainty of the traditional random transgenic mouse in the prior art are solved, and the animal model construction method provided by the invention has the characteristics of high gene editing efficiency and definite integration site;
the problem that the gene integration copy number of the traditional random transgenic mouse is not fixed in the prior art is solved, and the animal model construction method provided by the invention has the characteristic of fixed gene integration copy number;
the problem that the traditional random transgenic mouse in the prior art is high in time and money cost is solved, and the animal model construction method provided by the invention can reduce mouse customization cost and shorten construction period.
The invention adopts a CRISPR-Cas9 system, which mainly utilizes the double strand break of 3 bases at the upstream of an adjacent sequence (PAM sequence, NGG) of an original region of a target gene mediated by Cas9 nuclease under the guide of sgRNA, the HNH domain on Cas9 cuts single strand DNA complementary with the sgRNA, the Ruv C domain cuts non-complementary single strand, and the fixed point knock-in of a gene fragment is realized by homologous recombination Repair (Homology-Directed Repair, HDR) under the existence of a template strand. The system has the characteristics of high editing efficiency and targeting property, easy targeting, and more accurate and efficient experimental operation;
the invention uses repair donor containing mouse homologous sequence as homologous recombination repair template, and injects the repair donor and Cas9/sgRNA into mouse fertilized egg together, thus increasing the probability of homologous recombination repair and the probability of obtaining positive mouse, the mouse prepared by the method does not contain other exogenous sequences except the target sequence;
the ROSA26 site and the H11 site are respectively selected as specific insertion sites of a target gene E6/E7, the two sites both belong to safe regions, the fixed-point insertion of an exogenous gene cannot influence the expression of other genes, and the E6/E7 cannot influence the mutual expression;
the promoter-loxP-Stop-loxP-Kozak-HPV E6 or E7-screening marker-polyA sequence is embedded into a specific site at a fixed point, and the promoter and the target gene are separated by a loxP-Stop-loxP structure, so that the promoter cannot start the expression of the target gene under normal conditions. Only after mating with the tissue-specific Cre tool mouse, the target gene can be expressed in specific cells or tissues, and the method has better specificity and controllability compared with the conventional transgenic mouse model;
the invention realizes the insertion of single copy target gene, and the expression quantity is easier to predict; different screening markers are added in the targeting vector, and then the model can be flexibly detected through the markers;
the invention can reduce the customization cost of the gene knock-in mouse model and greatly shorten the research and development period;
the mouse model can be hybridized with any Cre mouse with a tissue-specific promoter or infected by Cre virus, so that the function of E6/E7 genes can be selectively regulated and controlled by individual specific tissues or specific time, and the finally obtained conditional overexpression E6/E7 mouse can be used for researching the influence of E6/E7 on the processes of inducing cell malignant transformation, tumor microenvironment regulation, immune escape and the like in the HPV infection process.
Drawings
FIG. 1 is a schematic diagram showing the overall strategy of gene editing in example 1 of the present invention.
Fig. 2 is an evaluation of sgRNA off-target effect in example 1 of the present invention.
FIG. 3 is a schematic view of a targeting vector in example 1 of the present invention.
FIG. 4 is a diagram showing the results of enzyme digestion verification of the targeting vector in example 1 of the present invention.
FIG. 5 is a schematic diagram of the design of primers for identifying PCR of a targeted mouse in example 1 of the present invention.
FIG. 6 is a diagram showing the results of PCR electrophoresis of the 5' homology arm of F1 mouse in example 1 of the present invention.
FIG. 7 is a diagram showing the results of PCR electrophoresis of the 3' homology arm of the F1 generation mouse in example 1 of the present invention.
FIG. 8 is a graph showing the sequencing alignment of the PCR products of the 5' homology arm of the F1 mouse in example 1.
FIG. 9 shows the sequencing alignment of the 3' homology arm PCR products of the F1 mouse in example 1.
FIG. 10 is a schematic diagram of alternative PCR identification primers for a target mouse in example 1 of the present invention.
FIG. 11 is a schematic diagram of the restriction enzyme and primer design for identifying the southern blot of the targeted mouse in example 1.
FIGS. 12A and 12B are graphs showing the results of southern blot analysis of the targeted mouse in example 1 of the present invention.
FIG. 13 is a schematic diagram showing the overall strategy for gene editing in example 2 of the present invention.
Fig. 14 is an evaluation of sgRNA off-target effect in example 2 of the present invention.
FIG. 15 is a schematic view of a targeting vector in example 2 of the present invention.
FIG. 16 is a diagram showing the results of enzyme digestion verification of the targeting vector in example 2 of the present invention.
FIG. 17 is a schematic diagram of the design of primers for identifying PCR of a target mouse in example 2 of the present invention.
FIG. 18 is a diagram showing the results of PCR electrophoresis of the 5' homology arm of F1 mouse in example 2 of the present invention.
FIG. 19 is a diagram showing the results of PCR electrophoresis of the 3' homology arm of F1 mouse in example 2 of the present invention.
FIG. 20 is a graph showing the sequencing alignment of the PCR products of the 5' homology arm of the F1 mouse in example 2 of the present invention.
FIG. 21 shows the sequencing alignment of the 3' homology arm PCR products of the F1 mouse in example 2 of the present invention.
FIG. 22 is a schematic diagram of alternative PCR identification primers for a target mouse in example 2 of the present invention.
Detailed Description
The technical solution of the present invention is further described below with reference to specific examples.
Specific example 1: construction of animal model for conditional overexpression of HPV16E6 at ROSA26 site
The HPV16E6 gene sequence is knocked in at the ROSA26 site of a mouse model by using a CRISPR-Cas9 gene editing technology, so that the conditional overexpression of HPV16E6 can be realized, and a mouse model of the conditional overexpression of HPV16E6 is established. The method comprises the following steps:
(1) sgRNA design and off-target effect assessment
sgRNA design for conditional overexpression of HPV16E6 at the ROSA26 site in C57BL/6 mice was performed, and candidate sgrnas were evaluated for off-target effects, as shown in fig. 2. The sgrna (formatting forward strandofgene) is obtained by screening:
CUCCAGUCUUUCUAGAAGAUGGG。
(2) incubating the obtained sgRNA and the Cas9 protein together to prepare a Cas9/sgRNA mixture;
(3) gene editing strategy and targeting vector construction
The overall strategy of gene editing is shown In figure 1, and for achieving the purpose of gene editing, as shown In figure 3, an In-Fusion cloning technology is utilized to construct a homologous recombination vector carrying a conditional overexpression E6 sequence, a recombination system used In the construction process is a Cre-LoxP induced expression system, and the vector comprises a gene segment of 'CAG-loxP-Stop-loxP-Kozak-HPV 16E6-3 FLAG-polyA'. The results of the vector restriction verification are shown in FIG. 4.
(4) Microinjection was performed with F0-generation mice
Co-injecting the targeting vector and the Cas9/sgRNA mixture into mouse fertilized eggs; and sending the fertilized eggs after microinjection back to the oviduct of the surrogate mouse, and screening out a target mouse, namely the F0 surrogate mouse, through gene identification after the mouse is born.
(5) F1 generation positive heterozygote mice
And (3) breeding the sexually mature positive F0 mice and wild mice to generate F1 generations, and finally obtaining positive heterozygote F1 generation mice after PCR and Southern verification to complete the mouse model construction capable of subculturing.
(6) F2 generation positive homozygous mice
Selfing the positive heterozygote F1 mouse, and screening to obtain the F2 positive homozygote mouse.
(7) The mouse genotype identification scheme comprises the following steps:
APCR detection and sequencing verification
Genomic DNA from mouse rat tail was extracted from E6 and identified by PCR after targeting in repair donor. As shown in FIG. 5, the primers ROSA26-seqF1/ROSA26-seqR1 are respectively positioned outside the 5 'homologous arm and in the repair donor fragment, and PCR products are generated by the amplification of the primers, so that the target donor is effectively inserted in the 5' of the mouse genome; ROSA26-seqF2/ROSA26-seqR2 are respectively positioned in the fragment of the repirandor and outside the 3 'homologous arm, and if the pair of primers is amplified to generate a PCR product, the target doror is effectively inserted in the 3' of the mouse genome. And the PCR products were sequenced and aligned using primers ROSA26-seqF3/ROSA26-seqR3, respectively.
PCR primer sequences:
primer name | Primer sequences |
F1 | 5’-AAAGATCGCTCTCCACGCCCTAG-3’ |
R1 | 5’-AGATGTACTGCCAAGTAGGAAAGTC-3’ |
F2 | 5’-CTGCTGTCCATTCCTTATTCCATAG-3’ |
R2 | 5’-CTGGAAATCAGGCTGCAAATCTC-3’ |
F3 | 5’-CACTTGCTCTCCCAAAGTCGCTC-3’ |
R3 | 5’-ATACTCCGAGGCGGATCACAA-3’ |
And (3) PCR reaction system:
and (3) PCR reaction conditions:
in this example, 3 positive F1 mice were obtained by PCR and sequencing verification, No. 2, No. 3, No. 4: the results of 5 'and 3' homology arm PCR identification electrophoresis are shown in FIGS. 6 and 7, respectively (number: F1 mouse number; WT: wild-type control); the 5 'and 3' homology arm PCR products were taken for sequencing and sequence alignment, respectively, and the results are shown in FIG. 8 and FIG. 9 for mouse No. 2.
Remarking:
1) the Taq DNA polymerase used was LongAmp Taq DNA polymerase (NEB M0323V).
2) Two controls used in PCR genotyping were:
controlling water: no DNA template was added.
Wild type control: 400ng of mouse genomic DNA.
If the DNA sample is of lower purity or it is desirable to shorten the PCR reaction time. Alternative primers as in figure 10 may be used.
Alternative PCR primer sequences:
primer name | Primer sequences |
F4 | 5’-AGATCTGCAAGCTAATTCCTGC-3’ |
R4 | 5’-TGCATAACTGTGGTAACTTTCTG-3’ |
Alternative PCR reaction system:
reaction assembly | Volume of |
MousetailgenomicDNA | 1μL |
Forwardprimer(10μM) | 1μL |
Reverseprimer(10μM) | 1μL |
PremixTaqPolymerase | 12.5μL |
ddH2O | 9.5μL |
Total | 25μL |
Alternative PCR reaction conditions:
b southern blot identification
The double-arm homologous recombination positive mice (No. 2-4) verified by PCR and sequencing are taken to extract rat tail DNA. As shown in FIG. 11, the DNA probe used for Southern blot analysis was selected while being cleaved with restriction enzymes BamHI or BstEII. As shown in FIGS. 12A and 12B, the detection results show that the three mouse DNA fragments can be hybridized with the designed probe, and the sizes of the products are in accordance with the expectation.
5' Probe primer sequence:
primer name | Primer sequences |
Forward | 5’-AAACGTGGAGTAGGCAATACCCAGG-3’ |
Reverse | 5’-AAAGAAGGGTCACCTCAGTCTCCCT-3’ |
3' Probe primer sequence:
primer name | Primer sequences |
Forward | 5’-TTCTGGGCAGGCTTAAAGGCTAAC-3’ |
Reverse | 5’-AGGAGCGGGAGAAATGGATATGAAG-3’ |
Specific example 2: construction of animal model for conditional overexpression of HPV16E7 at H11 site
The HPV16E7 gene sequence is knocked in at the site of H11 of a mouse model by using a CRISPR-Cas9 gene editing technology, so that the conditional overexpression of HPV16E7 can be realized, and a mouse model of the conditional overexpression of HPV16E7 is established. The method comprises the following steps:
(1) sgRNA design and off-target effect assessment
sgRNA design for conditional overexpression of HPV16E7 at the C57BL/6 mouse H11 site and off-target effect assessment of candidate sgrnas were performed as shown in fig. 14. Sgrna (formatting forward strand gene) was screened:
GAACACUAGUGCACUUAUCCUGG。
(2) incubating the obtained sgRNA and the Cas9 protein together to prepare a Cas9/sgRNA mixture;
(3) gene editing strategy and targeting vector construction
The overall strategy of gene editing is shown In FIG. 13, and for achieving the purpose of gene editing, as shown In FIG. 15, an In-Fusion cloning technology is used for constructing a homologous recombination vector carrying a conditional overexpression E7 sequence, a recombination system used In the construction process is a Cre-LoxP induced expression system, and the vector comprises
"CAG-loxP-Stop-loxP-Kozak-HPV 16E 7-HA-polyA" gene fragment. The results of the vector restriction verification are shown in FIG. 16.
(4) Microinjection was performed with F0-generation mice
Co-injecting the targeting vector and the Cas9/sgRNA mixture into mouse fertilized eggs; and sending the fertilized eggs after microinjection back to the oviduct of the surrogate mouse, and screening out a target mouse, namely the F0 surrogate mouse, through gene identification after the mouse is born.
(5) F1 generation positive heterozygote mice
And (3) breeding the sexually mature positive F0 mice and wild mice to generate F1 generations, and finally obtaining positive heterozygote F1 generation mice after PCR and Southern verification to complete the mouse model construction capable of subculturing. (6) F2 generation positive homozygous mice
Selfing the positive heterozygote F1 mouse, and screening to obtain the F2 positive homozygote mouse.
(7) The mouse genotype identification has two schemes of AB:
scheme A: PCR detection and sequencing verification
Genomic DNA from mouse rat tail was extracted from E7 and identified by PCR after targeting in repair donor. As shown in FIG. 17, primers H11-seqF1/H11-seqR1 are respectively located outside the 5 'homology arm and inside the repair donor fragment, and PCR products are generated by the amplification of the primers, which indicates that the target donor is effectively inserted in the 5' of the mouse genome; H11-seqF2/H11-seqR2 are respectively positioned in the fragment of the repair donor and outside the 3 'homology arm, and if the pair of primers is amplified to generate a PCR product, the target donor is effectively inserted in the 3' of the mouse genome. And sequencing and sequence alignment are carried out on the PCR products by using primers H11-seqF3/H11-seqR3 respectively.
PCR primer sequences:
primer name | Primer sequences |
F1 | 5’-GTACATCCACAGCATCTTCCAAG-3’ |
R1 | 5’-AGATGTACTGCCAAGTAGGAAAGTC-3’ |
F2 | 5’-GCATCTGACTTCTGGCTAATAAAG-3’ |
R2 | 5’-GCCTTGACCTAAGAGATGATGCGAC-3’ |
F3 | 5’-CTCTACTGGAGGAGGACAAACTG-3 |
R3 | 5’-GTCTTCCACCTTTCTTCAGTTAGC-3’ |
And (3) PCR reaction system:
reaction assembly | Volume of |
MousetailgenomicDNA | 2μL |
Forwardprimer(10μM) | 2μL |
Reverseprimer(10μM) | 2μL |
dNTPs(2.5mM) | 6μL |
5XLongAmpTaqReaction | 10μL |
LongAmpTaqDNAPolymerase | 2μL |
ddH2O | 26μL |
Total | 50μL |
And (3) PCR reaction conditions:
in this example, 3 positive F1 mice were obtained by PCR and sequencing verification, No. 9, 10, 11: the 5 'and 3' homology arm PCR identification electrophoresis results are shown in FIGS. 18 and 19, respectively, wherein the numbers: the mouse numbers of F1 generation; WT: wild type control); the 5 'and 3' homology arm PCR products were taken for sequencing and sequence alignment, respectively, and the results are shown in FIG. 20 and FIG. 21, using mouse No. 10 as an example.
Remarking:
1) the Taq DNA polymerase used was LongAmp Taq DNA polymerase (NEB M0323V).
2) Two controls used in PCR genotyping were:
controlling water: no DNA template was added.
Wild type control: 400ng of mouse genomic DNA.
If the DNA sample is not very pure or does not have sufficient PCR extension time, it may not be possible to amplify long fragment PCR products. Alternative primers as shown in FIG. 22 may be used.
Alternative PCR primer sequences:
primer name | Primer sequences |
F4 | 5’-GTGTGACTCTACGCTTCGGTTGTGC-3’ |
R4 | 5’-CTTTATTAGCCAGAAGTCAGATGC-3’ |
Alternative PCR reaction system:
reaction assembly | Volume of |
MousetailgenomicDNA | 1μL |
Forwardprimer(10μM) | 1μL |
Reverseprimer(10μM) | 1μL |
PremixTaqPolymerase | 12.5μL |
ddH2O | 9.5μL |
Total | 25μL |
Alternative PCR reaction conditions:
scheme B southern blot identification
Double-arm homologous recombination positive mice (No. 9-11) verified by PCR and sequencing are taken to extract rat tail DNA. The DNA probes used for Southern blot analysis were selected simultaneously by restriction with the restriction enzymes EcoRI or Ssspl. The detection result shows that the corresponding genome segments of the three mice subjected to DNA enzyme digestion can be hybridized with the designed probe, and the size of the product is in accordance with the expectation.
KI probe primer sequence:
primer name | Primer sequences |
Forward | 5’-TGCCCTGGCTCACAAATACCACT-3’ |
Reverse | 5’-TAGCCAACCTTTGTTCATGGCAGC-3’ |
Specific example 3: generation of conditional site-directed HPV16E 6/E7 double knock-in mice
Hybridization to obtain a conditional site-directed HPV16E 6/E7 double knock-in mouse method: mice homozygous for ROSA26 site conditional overexpression HPV16E6 constructed in example 1 were crossed with the mouse model homozygous for H11 site conditional overexpression HPV16E7 constructed in example 2 to obtain positive progeny of mice homozygous for conditional fixed-point HPV16E 6/E7 double knock-in. The heterozygous progeny is 100% homozygous and may not be genotyped.
The invention provides an animal model which can stably inherit and accord with the real pathological state of HPV infection by establishing a conditional fixed-point HPV E6/E7 double-transgenic mouse model through a transgenic technology. The model can be used for researching the HPV key oncogene E6/E7 in vivo function analysis, disease pathogenesis discussion, drug new target discovery, preclinical efficacy evaluation and the like, and has very important scientific significance and clinical value.
SEQUENCE LISTING
<110> Shanghai Coincident Biotechnology Co., Ltd
<120> construction method of mouse model of conditional site-directed double overexpression HPV E6/E7 gene
<130>JSP11912145
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Claims (9)
1. A method for constructing a mouse model of conditional site-specific dual overexpression HPV E6/E7 gene is characterized by comprising the following steps:
1. obtaining mice with conditional site-directed tapping of the ROSA26 site HPV E6 and H11 site-conditional site-directed tapping HPV E7:
(1) designing and obtaining sgRNA at mouse ROSA26 site and H11 site respectively according to the sequences of HPV E6 and E7 genes, and evaluating the off-target effect of candidate sequences;
(2) respectively incubating sgRNAs corresponding to the two sequences with Cas9 protein to respectively prepare Cas9/sgRNA mixtures;
(3) constructing a targeting vector, and constructing homologous recombination vectors carrying conditional overexpression E6 or E7 sequences respectively by utilizing In-Fusion cloning technology, wherein a recombination system used In the construction process is a Cre-LoxP induced expression system, and the vectors comprise 'start-loxP-Stop-loxP-Kozak-HPV E6 or E7-screening marker-polyA' gene segments;
(4) microinjection and F0 generation mice, respectively, and the targeting vector and the Cas9/sgRNA mixture are injected into fertilized eggs of the mice together; sending the fertilized eggs subjected to microinjection back to the oviduct of a surrogate mouse, and after the mouse is born, screening out a target mouse through gene identification, namely an F0 generation mouse, including an F0 generation mouse of which the site of ROSA26 is knocked into HPV E6 at a fixed point and an F0 generation mouse of which the site of H11 is knocked into HPV E7 at a fixed point;
(5) obtaining mice of F1 generation, breeding the sexually mature positive F0 generation mice with wild mice to generate F1 generation, and obtaining F1 generation positive heterozygote mice after PCR and Southern verification, wherein the F1 generation positive heterozygote mice comprise F1 generation mice of ROSA26 site fixed point knock-in HPV E6 and F1 generation mice of H11 site fixed point knock-in HPV E7;
(6) selfing the F1 generation positive heterozygote mouse with the site of ROSA26 knocked-in HPV E6 and the F1 generation positive heterozygote mouse with the site of H11 knocked-in HPV E7 respectively, and screening to obtain an F2 generation positive homozygote mouse;
2. obtaining a conditional site-directed HPV E6/E7 double knock-in model mouse:
the mouse with the ROSA26 site fixed point knocked-in HPV E6F 2 generation positive homozygote is hybridized with the mouse with the H11 site fixed point knocked-in HPV E7F2 generation positive homozygote to obtain a conditional fixed point HPV E6/E7 double-knock-in model mouse.
2. The method of constructing a mouse model of conditional overexpression of the HPV E6 gene at the ROSA26 site of claim 1, wherein:
the sequences of sgrnas conditionally overexpressing HPV16E6 are:
CUCCAGUCUUUCUAGAAGAUGGG,
the sequences of sgrnas conditionally overexpressing HPV16E7 are:
GAACACUAGUGCACUUAUCCUGG。
3. the method of constructing a mouse model of conditionally overexpressing HPV E6 at the ROSA26 site of claim 1, further comprising:
mouse genotype identification: by adopting PCR detection or sequencing verification,
extracting genome DNA of an E6 mouse rat tail, and performing PCR identification after the target of the repair donor is in the target, wherein primers ROSA26-seqF1/ROSA26-seqR1 are respectively positioned outside a 5 'homology arm and in a repair donor fragment, and if a PCR product is generated by amplification of the pair of primers, the target donor is effectively inserted in the 5' genome of the mouse; ROSA26-seqF2/ROSA26-seqR2 are respectively positioned in the fragment of the repair donor and outside the 3 'homology arm, if the pair of primers are amplified to generate a PCR product, the target donor is effectively inserted into the 3' of the mouse genome;
e7 mouse rat tail genomic DNA was extracted and subjected to PCR identification after targeting with repair donor. As shown in FIG. 19, primers H11-seqF1/H11-seqR1 are respectively located outside the 5 'homology arm and in the repair donor fragment, and PCR products are generated by the amplification of the primers, which indicates that the target donor is effectively inserted in the 5' of the mouse genome; H11-seqF2/H11-seqR2 are respectively positioned in the fragment of the repair donor and outside the 3 'homology arm, and if the pair of primers is amplified to generate a PCR product, the target donor is effectively inserted in the 3' of the mouse genome.
5. the method of constructing a mouse model of conditional overexpression of the HPV E6 gene at the ROSA26 site of claim 4, wherein:
if the purity of the DNA sample is low or the PCR reaction time is desired to be shortened, alternative PCR primers are adopted, and PCR products with shorter fragments can be obtained, wherein the sequences of the alternative PCR primers are respectively as follows:
e6 mice:
e7 mice:
。
6. The method of constructing a mouse model of conditional overexpression of the HPV E6 gene at the ROSA26 site of claim 5, wherein:
alternative PCR reaction system:
。
8. the method of constructing a mouse model of conditionally overexpressing HPV E6 at the ROSA26 site of claim 1, further comprising:
mouse genotype identification: and (3) identifying by using Southern blot:
taking the double-arm homologous recombination positive mouse verified by PCR and sequencing, extracting rat tail DNA, carrying out enzyme digestion by restriction enzyme BamHI or BstEII, simultaneously selecting a DNA probe for Southern blot analysis,
e6 mouse 5' probe primer sequence:
e6 mouse 3' probe primer sequence:
e7 mouse KI probe primer sequence:
。
9. The method of constructing a mouse model of conditionally overexpressing HPV E6 at the ROSA26 site of claim 1, further comprising:
crossing the fixed point HPV E6 single knock-in mouse, the fixed point HPV E7 single knock-in mouse or the fixed point HPV E6/E7 double knock-in model mouse with Cre mouse with tissue specific promoter or infecting with Cre virus.
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CN112997966A (en) * | 2021-03-08 | 2021-06-22 | 国家卫生健康委科学技术研究所 | Mouse model knocking-in miRNA-125a based on CRISPR/Cas9 technology and construction method |
CN115074368A (en) * | 2022-06-09 | 2022-09-20 | 澳门科技大学 | Construction and application of drug-resistant rheumatoid arthritis animal model |
CN115074368B (en) * | 2022-06-09 | 2023-08-08 | 澳门科技大学 | Construction and application of drug-resistant rheumatoid arthritis animal model |
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