CN116656681B - Construction method and application of CD34 transgenic mode mouse - Google Patents
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
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Abstract
The invention relates to the technical field of bioengineering, and discloses a construction method and application of a CD34 transgenic mode mouse, wherein the construction method comprises the following steps: s1, designing and screening to obtain sgRNA according to a target site of a rat tail target sequence, and constructing a Cas9/sgRNA plasmid vector by using the sgRNA; s2, knocking the marker gene sequence into a CD34 gene sequence, and constructing a recombinant targeting vector; and S3, introducing the Cas9/sgRNA plasmid vector and the recombinant targeting vector into mice, and screening after passage to obtain F1 generation recombinant positive mice. The construction method is simple, the success rate is high, and the offspring mice obtained by mating the CD34 transgenic mode mice obtained by the method with other mice can specifically track CD34 positive cell changes, so that the method has important significance in promoting early lymphopoiesis stem cell/progenitor cell research.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a construction method and application of a CD34 transgenic mode mouse.
Background
CD34 molecules are highly glycosylated type I transmembrane glycoproteins, whose structure comprises extracellular, transmembrane, cytoplasmic domain 3 portions, which are selectively expressed on the surface of human and other mammalian hematopoietic stem/progenitor cells, and thus CD34 molecules are commonly used to label early lymphohematopoietic stem/progenitor cells, as well as resident progenitor cells of various tissues (e.g., fat, dermis, skeletal muscle, etc.).
In the related art, the function of CD34 is very complex, and research shows that CD34 molecules play an important role in mediating the cell-cell adhesion, can participate in the transportation, the colonization and the inflammatory reaction of hematopoietic stem cells, and also have the functions of maintaining the stable internal environment, supplementing T, B cells of peripheral lymph nodes and the like. Currently, the expression of CD34 in tissues is usually studied by using an immunofluorescent staining method in related researches, however, the method can only be used for judging the real-time expression of CD34 in tissues, and the follow-up division and differentiation of CD34 positive cells cannot be tracked.
Thus, there is a need to find a new method that enables specific tracking of changes in CD34 positive cells.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a construction method and application of a CD34 transgenic mode mouse, which can realize specific tracking of CD34 positive cell changes.
The invention also provides sgRNA for editing the mouse CD34 gene.
The invention also provides a CRISPR/Cas9 system expression vector targeting the region between the mouse CD34 gene start codon and the exon 1.
The invention also provides a recombinant targeting vector.
The invention also provides a CRISPR/Cas9 system for knocking in a marker gene at a fixed point of a mouse CD34 gene.
The invention also provides a construction method of the CD34 transgenic mode mouse.
The invention also provides application of the sgRNA in specific recognition and targeted editing of a mouse CD34 gene or construction of a mouse with a CD34 transgenic mode.
The invention also provides application of the construction method of the CD34 transgenic mode mouse in obtaining the mouse with the function of specifically tracking the CD34 positive cells.
In a first aspect of the invention there is provided an sgRNA for editing the murine CD34 gene, said sgRNA having the nucleotide sequence shown in SEQ ID NO.4 or SEQ ID NO. 5.
The sgRNA provided by the embodiment of the invention has at least the following beneficial effects: the sgRNA is designed and screened according to the target site of the rat tail target sequence, and has high activity and low off-target efficiency.
In some embodiments of the invention, the nucleotide sequence of the sgRNA is shown as SEQ ID No. 5.
In some embodiments of the invention, the murine CD34 gene is located on the positive strand of mouse chromosome 1, full length 22.5kb,Gene ID:12490.
In some embodiments of the invention, the mouse is a C57BL/6N mouse.
In a second aspect of the invention, there is provided a CRISPR/Cas9 system expression vector targeting the region between the start codon and exon1 of the murine CD34 gene, said expression vector consisting of the sgRNA and the backbone vector pCS described above.
In some embodiments of the invention, the method of constructing a CRISPR/Cas9 system expression vector comprises: the sgRNA sequence (shown as SEQ ID NO.4 or SEQ ID NO. 5) is connected to a plasmid vector with a promoter, an oligonucleotide is synthesized after in vitro transcription, and then the oligonucleotide is connected to a pCS vector, thus obtaining the Cas9/sgRNA system expression vector.
In some embodiments of the invention, the promoter comprises a T7 promoter.
In a third aspect of the invention, a recombinant targeting vector is provided, wherein the recombinant targeting vector comprises a homologous arm at the 5 'end of a mouse CD34 gene, the mouse CD34 gene, a CrePR1-P2A-tdTomato-WPRE-pA sequence and a homologous arm at the 3' end of the mouse CD34 gene;
wherein the CrePR1-P2A-tdTomato-WPRE-pA sequence is shown as SEQ ID NO. 14;
the knockin site of the CrePR1-P2A-tdTomato-WPRE-pA sequence is between the start codon of the mouse CD34 gene and the exon 1.
The experimental verification shows that the invention has minimal influence on CD34 gene expression when the knock-in site is between the start codon of the mouse CD34 gene and the exon 1.
In some embodiments of the invention, the backbone vector of the recombinant targeting vector is a PUC19 vector.
In some embodiments of the invention, the start codon of the murine CD34 gene is ATG.
In some embodiments of the invention, the 5 '-end homology arm of the murine CD34 gene is 1.3-2 kb in length and the 3' -end homology arm of the murine CD34 gene is 1.3-2 kb in length.
Wherein the 5' homology arm sequence is shown in SEQ ID NO. 21:
CATAAAAAGTATGAGCAAAATGTAGGTACCAGAAGAAAATCCACTTTTGAAACTGAAGAATTAGGTGTAATATTTCCTTCTTAGTTCATATTCCAAGGACCAGAATTTAATTTTAAAAGTACATTTTGCAATCCTTTCTGACCCTACCTCCAGGTTCTCCATCACAATATGCCAACTGTACTTTGACACAAACTCACTTAATGTCGTTGTATTCAATGAGCCTCTGCCTTTATTATGATGAAAACCTCCGCTTCTTGTTTTAATGGTGTGATATGAAACAGGGTAAGAGTATAGATTTGAGGACTGATACTCTTCTTCTCAACCCCAAGGTATATTAGGCCCAGGGTAGGCAACACAAACAAATGTACTTTCTTCGTGGAGGTAGATCTTCAGAGAAATAAATACAGAAATTTATCCAGGGCGTTTGTCTGATCTTTCTTTCTCCCTTTAGGTTATGTGACCAGACATTATGGGAAAACTAGGGCTAAGTATTTACAGTCATCATCACAGACCCTTCTGTGCCTTATCTCAATTTCTACCTGCTGCTATATGCCTTGTCTTGCTTCTCTCTCCTCCTGCCTTGATCTTCTGACCTGTGACGAGTCAGAATCCTGCAATCCAGCGCATCAACATGCAGCACCATTGCGTGGAGAAGCACAAGGAGTGCTAGAAGGTCTGTTAGGTAGGGCAGGGTAAGAATCTAAGAAATCTTTTGTCTGAAGGGAGGGCAAGAAAAGCTCCCATAGAATCAGGAAACTTAAATCTAGGATTGAAAAGGGAACATTGGAGAAAAACCATGTACTATGGAGTATCTATATTATCTCTATGGTATTGACAATTGCTATGACCCAACTAGCTAAGGAAATATGGATGATCACAGGAAAGTTAATACAAATTCTGTTGATTTTGCTGGAAAATGAGTATAAAAGAGAGATGGTAGGATAGCAGATGGTCTGGACTGTGAAAAGGCTATTAGTGTCCTGTTTGTGTCCTGCTCAATTTTAACATGCAGCACAGATTCCACCGTGACTATCTCTAAAGGTGTCTTGGAATTGTGTTGTTTCCTGCTTAGCTGAAAGATCATACTCCTGTAAACTCAGGCGTACACAGTGGCTTCCTGCCACACTTAAAACTTGAAAATAAAGAAATCAGAAAGGAGAAATTTGACCCTGCCGAGAGGCAGCCAAGATGACACACGGTTAAAAGTGAAGTAGGAACTACGAGAGGGGCTGGCCTCACCAAGACGCAACAGGGAGGGGATAAGCCAGCATCCCCCACCCACTCCGGACAGGGAGCAGGGGAGGAGAGCCAATATCCCCCACCCCTGCGCAGGGCGGAGGAGCGCGTCCCGCGCCGGGCCGCCTCCTGCACCGAGCGCATCTCCGGAGCGGTACAG(SEQ ID NO.21)。
wherein the 3' homology arm sequence is shown in SEQ ID NO. 22:
CAGGTCCACAGGGACACGCGCGCGGGGCTCCTGCTGCCATGGCGCTGGGTAGCTCTCTGCCTGATGAGTCTGCTGCGTGAGTATTAACAGCTGAGGGCCAAGGGACCAGGCTATGGGCCGGGAGGAGGGAAGACGCCGGCCACTAACCTCACCTGGGAATCCTGCGCTCCAGCACGCAGAAGTGAGCTTACTCAGGTAGTGCTGGGGGACGCCAGCTCCTCTGCGTGGCTGGAGGTGCAAAGCTCAAAGTTGAGGCTAGAATGCAGACAGAAGGAAGGAAAAGTTCTGTAACTCTCCTTAATTGAGGAAGCAAGGACCCAGAGTGGTCCAGAGAAGGGTCCAGGCACCCATTGTTCCCAGGACTTGGACATAAAGAATTCATGAGGGTAGCCATTTCCAGTAAGGGCCAGGAGACCTTTAAGGAGGACTGAGGGACATTAGTGGAAGAGTCTGGAATAAAGAACAGGCAGAGAGGAAAAAAAGGACAATTTTAAATTTCAGATTAGAAAGCTAGAGTCGCCCCTGCAGAAACCATGCAGATGCTAGTAGGGAGGTGGTTTGGTGTACACACCAAGTCCTTCTGCTCACCAGTCTTCCCCGTGCACAAACATACATTCATTGCTAGTTGGAATTTTCTTGCTCTGCTTCCCTTCAGATTCTAAAGCTAGTGGACAGGCCAAGACACCTCCTGCTGCCCACGCATACTCTTACTTCTTAAGTTCTTCCCCTCTTAGGTTGGCAAAAAATAGTATTCACTCAGTCTATAGCAAACTTTATTGCACTGGAGGGTGTTGTTCTTCATCCCAACTCATGTCCTTTGAGGTTTTCAGGGTTGTGAATTACTCATTTTGCTTTTAACCCTTGGGGCTCCTAAATCTTCATTGTATATCTTAAAGGACACTCAACATGTAGAGTAGCCTGGATCTACCCACAAGACCTCTTTTCTCCCAGTCTTCTGCTCCATTGCCTTTGGGTCTATCCCAAAGAGAGCTGGCAGAGGAGAAGTTGAATTAGGTTCTGGGTTCTCCTGATTGGCAGCGCTAGACATGTCCATTTTGATGGAGTAAAACTTGCTTGGACAAAAATGCCCAGAATGATGCTACACTGTGCACGGGTAGGTGACCACGGGCTGGAGTCCTCATCAGATGCTCTGTACTTACTCTTTTGGTCACTCAGCTCAGCACTGACCCAAGTCTGGGTGTAGTGCTGTCTTTTCTTATTTCCTCAGCTGGCCAGTATCAGCCTTGTCTTGGGCTGAATGGGGCAGGGTGGAGAGAGCAAAAAAACCTCTGCAAACCTTGGGGAAATATGCTGAAGATATCAGTGTCTCAACTTTAGGGTAAAGGACAGGGCAGGGGCCACCATTCCCAACCCAGCTAGACTTCATAAGCCGGCCAAGG(SEQ ID NO.22)。
in a fourth aspect of the invention, there is provided a CRISPR/Cas9 system for site-directed knock-in of a marker gene at a murine CD34 gene, said CRISPR/Cas9 system comprising a CRISPR/Cas9 system expression vector and a recombinant targeting vector as described above.
In some embodiments of the present invention,
in a fifth aspect of the present invention, there is provided a method for constructing a CD34 transgenic mode mouse, comprising the steps of:
introducing the CRISPR/Cas9 system expression vector and the recombinant targeting vector into fertilized eggs of mice, screening F0-generation positive mice, hybridizing the F0-generation positive mice with wild mice, and screening F1-generation recombinant positive mice to obtain the CD34 transgenic mode mice.
The construction method according to the embodiment of the invention has at least the following beneficial effects:
(1) According to the invention, firstly, a sRNA is designed to construct a Cas9/sgRNA plasmid, a CrePR1-P2A-tdTomato-WPRE-pA is knocked in at a fixed point in a CD34 gene to construct a recombinant targeting vector, then the Cas9/sgRNA plasmid and the recombinant targeting vector are injected into fertilized eggs of mice to develop so as to obtain F0 generation mice, then the F0 generation mice with positive genotypes are identified to be mated with wild type mice to obtain positive F1 generation mice, the F1 generation positive mice which are correctly recombined and are not randomly inserted are identified to be CD34 transgenic mouse models, and after the mouse models are mated with other related system mice, the obtained offspring mice can realize the functions of specifically tracking the change of CD34 positive cells, expressing or knocking out target genes in the CD34 positive cells, and the like, so that the development of the transgenic mouse models is greatly promoted.
(2) The construction method of the CD34 transgenic mode mouse is simple, the success rate is high, and the progeny mice obtained after the CD34 transgenic mode mouse is mated with other mice can specifically track the CD34 positive cell change.
In some embodiments of the invention, the primers for screening F0 positive mice comprise a CD34-L-GTF and CD34-L-GTR primer set, and a CD34-R-GTF and CD34-R-GTR primer set; wherein,
the nucleotide sequence of the CD34-L-GTF is shown as SEQ ID NO. 15;
the nucleotide sequence of the CD34-L-GTR is shown as SEQ ID NO. 16;
the nucleotide sequence of the CD34-R-GTF is shown as SEQ ID NO. 17;
the nucleotide sequence of the CD34-R-GTR is shown as SEQ ID NO. 18.
In the invention, the stripe length amplified by adopting the CD34-L-GTF and CD34-L-GTR primer sets is 5076bp, and the mice with the stripe length amplified by adopting the CD34-R-GTF and CD34-R-GTR primer sets are F0 generation positive mice, otherwise, F0 generation negative mice are identified.
In some embodiments of the invention, screening F1 generation recombinant positive mice comprises PCR screening and/or Southern blot screening.
Preferably, the PCR-screened primers include CD34-L-GTF and CD34-L-GTR primer sets, and CD34-R-GTF and CD34-R-GTR primer sets; wherein,
the nucleotide sequence of the CD34-L-GTF is shown as SEQ ID NO. 15;
the nucleotide sequence of the CD34-L-GTR is shown as SEQ ID NO. 16;
the nucleotide sequence of the CD34-R-GTF is shown as SEQ ID NO. 17;
the nucleotide sequence of the CD34-R-GTR is shown as SEQ ID NO. 18.
In some embodiments of the invention, the amplification procedure of the PCR is: the denaturation temperature is 94 ℃ and the denaturation is carried out for 2min; then 15 cycles were performed in which the DNA was denatured at 98℃for 10s, renatured at 67℃at a decrease of 0.7℃per cycle for 30s, and extended at 68℃at a rate of 1 kb/min; further 25 cycles were performed in which the DNA was denatured at 98℃for 30s, renatured at 57℃for 30s, and extended at 68℃at a rate of 1 kb/min; maintaining at 68deg.C for 10min, cooling to 4deg.C, and maintaining.
In some embodiments of the invention, the probes screened by the Southern blot method include 3' Probe and Cre Probe;
wherein the nucleotide sequence of the 3' probe is shown as SEQ ID NO. 20;
the nucleotide sequence of the Cre Probe is shown as SEQ ID NO. 19.
In the present invention, the 3' probe is used to detect whether correct recombination occurs, and if so, both wild-type and mutant bands will occur; if not properly recombined, only wild type bands will appear. The Cre Probe is used for detecting whether random insertion is contained or not, and if correct recombination occurs, only a target strip appears; if random insertion is present, multiple stripes will occur.
In some embodiments of the invention, the method of introducing comprises microinjection.
In some embodiments of the invention, the mouse is a C57BL/6N mouse.
In a sixth aspect of the invention, there is provided the use of the sgrnas described above for specific recognition and targeted editing of the murine CD34 gene or construction of a CD34 transgenic mode mouse.
In a seventh aspect, the invention provides an application of the construction method of the CD34 transgenic mode mouse in obtaining a mouse with the function of specifically tracking CD34 positive cells.
After the CD34 transgenic mode mice obtained by the construction method disclosed by the invention are mated with other mice of related systems, the obtained offspring mice can realize the functions of specifically tracking the change of CD34 positive cells, expressing or knocking out target genes in the CD34 positive cells and the like, and greatly promote the development of a transgenic mouse model.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a graph showing the results of the detection of sgRNA activity according to the example of the present invention.
FIG. 2 is an electrophoresis diagram of sgRNA2 according to an embodiment of the present invention.
FIG. 3 is a plasmid map of Cas9/sgRNA of an embodiment of the present invention.
FIG. 4 is a schematic diagram showing a targeting strategy for inserting CrePR1-P2A-tdTomato-WPRE-pA between CD34 gene initiation codon (ATG) and Exon1 (Exon 1) at fixed point according to an embodiment of the present invention.
FIG. 5 is a map of a targeting vector according to an embodiment of the present invention.
FIG. 6 is a graph showing the result of identifying 5076bp of the amplified product of the F0-generation mouse according to the present invention.
FIG. 7 is a graph showing the result of identifying 4781bp of the amplified product of the F0-generation mouse according to the present invention.
FIG. 8 is a graph showing the result of identifying 5076bp of the amplified product of the F1-generation mouse according to the present invention.
FIG. 9 is a graph showing the result of identifying 4781bp of the amplified product of the F1-generation mouse according to the present invention.
FIG. 10 is a graph showing the result of Southern blot detection of F1 mice in the present invention.
FIG. 11 is a fluorescence micrograph of a skin tissue section of a CD34 transgenic mouse of example 8W according to the present invention, wherein the length of the scale bar is 50. Mu.m.
FIG. 12 is a fluorescence micrograph of a back skin section of an example 8W CD34 mTMG mouse of the present invention, wherein the length of the scale bar is 50. Mu.m.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
In the description of the present invention, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In a specific embodiment of the invention, the CD34 gene is on chromosome 1 positive strand, full length 22.5kb,Gene ID:12490.
Example 1 acquisition and validation of target sequences of interest
And designing a primer sequence according to the CD34 gene information, and carrying out PCR amplification and sequencing verification on the rat tail target site sequence of the C57BL/6N mouse, wherein the primer sequence of the PCR amplification is shown in table 1.
Table 1: primer sequences
The PCR amplification system is shown in Table 2.
Table 2: amplification system
| Reagent(s) | Volume (20 mu L) |
| 2×Taq Plus Master Mix II(dye plus) | 10 |
| CD34-MSD-F(10μmol/μL) | 2 |
| CD34-MSD-R(10μmol/μL) | 2 |
| DNA template | 1 |
| ddH 2 O | 5 |
The PCR amplification procedure is shown in Table 3.
Table 3: amplification procedure
The amplified PCR product is purified and sequenced, and the result shows that the length of the rat tail target site sequence of the C57BL/6N mouse is 532bp, and specific sequence information is as follows:
GCATCAAGCTTGGTACCGATACAGTGGCTTCCTGCCACACTTAAAACTTGAAAATAAAGAAATCAGAAAGGAGAAATTTGACCCTGCCGAGAGGCAGCCAAGATGACACACGGTTAAAAGTGAAGTAGGAACTACGAGAGGGGCTGGCCTCACCAAGACGCAACAGGGAGGGGATAAGCCAGCATCCCCCACCCACTCCGGACAGGGAGCAGGGGAGGAGAGCCAATATCCCCCACCCCTGCGCAGGGCGGAGGAGCGCGTCCCGCGCCGGGCCGCCTCCTGCACCGAGCGCATCTCCGGAGCGGTACAGGAGAATGCAGGTCCACAGGGACACGCGCGCGGGGCTCCTGCTGCCATGGCGCTGGGTAGCTCTCTGCCTGATGAGTCTGCTGCGTGAGTATTAACAGCTGAGGGCCAAGGGACCAGGCTATGGGCCGGGAGGAGGGAAGACGCCGGCCACTAACCTCACCTGGGAATCCTGCGCTCCAGCACGCAGAAGTGAGCTTACTCAGATCATCCTCCACGATTAAGT(SEQ ID NO.3)。
after alignment, the results indicate that it is identical to the expected sequence.
Example 2 design and construction of CRISPR/sgRNA
1. Design and screening of sgrnas
The gRNA target sequence determines its targeting specificity and efficiency of inducing Cas9 cleavage of the gene of interest. The higher the efficiency with which Cas9 cleaves the gene of interest, the higher the efficiency with which homologous recombination occurs. Therefore, efficient specific target sequence selection and design is a prerequisite for successful construction of mouse models. Based on the design principle of sgrnas, 10 sgrnas were designed in total in the target site region of the C57BL/6N rat tail target sequence, and each sequence was tested for activity by activity detection, wherein the 10 sgrnas were designed as shown in table 4.
Table 4: sgRNA sequence information Table
| Sequence name | Sequence (5 '-3') | Sequence numbering |
| sgRNA1 | GGTCCACAGGGACACGCGCGCGG | SEQ ID NO.4 |
| sgRNA2 | GCGCATCTCCGGAGCGGTACAGG | SEQ ID NO.5 |
| sgRNA3 | TGCATTCTCCTGTACCGCTCCGG | SEQ ID NO.6 |
| sgRNA4 | GGAGCGGTACAGGAGAATGCAGG | SEQ ID NO.7 |
| sgRNA5 | TACCGCTCCGGAGATGCGCTCGG | SEQ ID NO.8 |
| sgRNA6 | GATGCGCTCGGTGCAGGAGGCGG | SEQ ID NO.9 |
| sgRNA7 | TCCTGCACCGAGCGCATCTCCGG | SEQ ID NO.10 |
| sgRNA8 | GCGCGGGGCTCCTGCTGCCATGG | SEQ ID NO.11 |
| sgRNA9 | CGGAGGAGCGCGTCCCGCGCCGG | SEQ ID NO.12 |
| sgRNA10 | TGCAGGAGGCGGCCCGGCGCGGG | SEQ ID NO.13 |
Activity detection method CRISPR/Cas9 Activity detection method-UCA independently developed with reference to Baioser diagram TM Mode(s).
The results of the activity detection are shown in FIG. 1, and the results show that the activity of the sgRNA2 sequence in the 10 sgRNA sequences is highest, so that the sgRNA2 sequence is selected for subsequent experiments.
2. RNA preparation of sgRNA
The sgRNA2 sequence with the highest activity is connected to a plasmid vector with a T7 promoter and is subjected to in vitro transcription, so that RNA capable of being subjected to microinjection is obtained, and an electrophoresis chart of the sgRNA2 is shown in FIG. 2 and is consistent with the expected result.
3. Construction of Cas9/sgRNA plasmids
The primer is prepared by synthesizing oligonucleotide (oligos) from the sgRNA2 sequence capable of microinjection, connecting the oligonucleotide to pCS vector by annealing polymerization, transferring the converted connection product to sample for sequencing and verifying correctness, and obtaining the Cas9/sgRNA plasmid vector, wherein the map is shown in figure 3.
EXAMPLE 3 construction of targeting vector
The schematic diagram of the targeting strategy of the invention is shown in fig. 4, which specifically comprises the following steps:
1. gene knockout mice were prepared using the EGE system developed by the baioser corporation based on CRISPR/Cas 9.
2. The targeting vector is obtained by inserting the CrePR1-P2A-tdTomato-WPRE-pA sequence between the initiation codon (ATG) of the CD34 gene and the Exon1 (Exon 1) in a fixed point manner, wherein the specific information of the CrePR1-P2A-tdTomato-WPRE-pA sequence is as follows:
TGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGATAAAAAGTTCAATAAAGTCAACAACTTCATCTGTACTGCATGGTGAGCAAGGGCGAGGAGGTCATCAAAGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCATGTACGGCTCCAAGGCGTACGTGAAGCACCCCGCCGACATCCCCGATTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCACGCTGATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACCAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGACCATCTACATGGCCAAGAAGCCCGTGCAACTGCCCGGCTACTACTACGTGGACACCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCTCCGAGGGCCGCCACCACCTGTTCCTGGGGCATGGCACCGGCAGCACCGGCAGCGGCAGCTCCGGCACCGCCTCCTCCGAGGACAACAACATGGCCGTCATCAAAGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCATGTACGGCTCCAAGGCGTACGTGAAGCACCCCGCCGACATCCCCGATTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCACGCTGATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACCAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGACCATCTACATGGCCAAGAAGCCCGTGCAACTGCCCGGCTACTACTACGTGGACACCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCTCCGAGGGCCGCCACCACCTGTTCCTGTACGGCATGGACGAGCTGTACAAGTGAAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG(SEQ ID NO.14)。
3. the result of enzyme digestion identification and sequencing of the targeting vector shows that the targeting vector contains a CrePR1-P2A-tdTomato-WPRE-pA fragment, and a 5 'homology arm and a 3' homology arm which are both about 2kb in length, and the sequence information is consistent with the expected sequence, and the construction of the targeting vector is completed, and the specific map of the targeting vector is shown in figure 5.
Example 4F0 Generation mice acquisition and identification
1. F0 mice acquisition
The Cas9/sgRNA plasmid obtained in example 2 and the targeting vector obtained in example 3 were injected into fertilized eggs of C57BL/6N mice by microinjection to obtain transfected fertilized eggs, and the transfected fertilized eggs were transplanted into female mice to give birth to the mice, and the F0 mice after injection were born as shown in table 5 below:
table 5: birth Condition table of F0 mice
| Conception date | Strain of strain | Number of transfected fertilized eggs | Date of production | Birth number of young animals |
| 2018/08/21 | C57BL/6N | 208 | 2018/09/09 | 9 |
| 2018/09/18 | C57BL/6N | 183 | 2018/10/07 | 23 |
| 2019/01/03 | C57BL/6N | 260 | 2019/01/22 | 27 |
2. Identification of F0 mouse genotype
The F0 mice obtained are chimeric due to the rapid cleavage rate in the early embryo. Therefore, the F0 genotype obtained by genotyping the tail of the F0 mouse is only used as a reference, and is not necessarily a heritable gene mutant type, and the heritable genotype can be determined after the genotyping of the F1 mouse.
Designing primers for genotype detection, taking an F0 chimeric mouse as an experimental group, taking a wild type (wild type) C57BL/6N mouse as a control group, and respectively extracting rat tail genome DNA (deoxyribonucleic acid) of the mice for PCR (polymerase chain reaction) verification, wherein the sequences of the primers for genotype detection are shown in table 6.
Table 6: primer sequence information for genotype detection
The PCR amplification system is shown in Table 7.
Table 7: amplification system
| Reagent(s) | Volume (20 mu L) |
| 2x Taq Plus Master Mix II(dye plus) | 10 |
| Primer F (10. Mu. Mol/. Mu.L) | 2 |
| Primer R (10. Mu. Mol/. Mu.L) | 2 |
| DNA template | 1 |
| ddH 2 O | 5 |
The PCR amplification procedure was: the denaturation temperature is 94 ℃ and the denaturation is carried out for 2min; then 15 cycles were performed in which the DNA was denatured at 98℃for 10s, renatured at 67℃at a decrease of 0.7℃per cycle for 30s, and extended at 68℃at a rate of 1 kb/min; further 25 cycles were performed in which the DNA was denatured at 98℃for 30s, renatured at 57℃for 30s, and extended at 68℃at a rate of 1 kb/min; maintaining at 68deg.C for 10min, and cooling to 4deg.C.
Gel electrophoresis is carried out on the amplified products, wherein the electrophoresis result of the amplified products of the CD34-L-GTF/CD34-L-GTR primers is shown in figure 6, and the electrophoresis result of the amplified products of the CD34-R-GTF/CD34-R-GTR primers is shown in figure 7, and the result shows that 11 PCR positive F0 mice and 1 suspected positive F0 mice are screened out, wherein the serial numbers of the PCR positive F0 mice are E3Z3-006, E3Z3-015, E3Z3-016, E3Z3-040, E3Z3-042, E3Z3-043, E3Z3-051, E3Z3-057, E3Z3-048, E3Z3-054 and E3Z3-055 respectively; the suspected positive F0 mice are numbered E3Z3-034.
Example 5 genotyping and southern blot detection of F1 mice
1. Obtaining F1-generation mice
Part of the genotypes of example 4 were identified as positive F0 mice E3Z3-006, E3Z3-016, E3Z3-040, E3Z3-042, E3Z3-043 and E3Z3-051 were mated with Wild type mice (Wild type, WT) to obtain F1 mice with stable genotypes, and mating results are shown in Table 8:
table 8: mating results table of F0 generation mice and wild type mice
| Numbering device | Conception date | Date of production | Birth number of young animals |
| E3Z3-006 | 2018/10/22 | 2018/11/12 | 7 |
| E3Z3-016 | 2018/11/20 | 2018/12/09 | - |
| E3Z3-042 | 2019/03/05 | 201903/24 | 19 |
| E3Z3-040 | 2019/03/13 | 2019/04/01 | 33 |
| E3Z3-043 | 2019/03/13 | 2019/04/01 | 44 |
| E3Z3-051 | 2019/03/13 | 2019/04/01 | 27 |
2. Identification of F1 Generation mice genotype
Primers for genotype detection were designed (see Table 6 specifically), F1 mice were used as experimental groups, wild type (wild type) C57BL/6N mice were used as control groups, and their rat tail DNA was extracted for PCR verification, wherein the PCR amplification system is shown in Table 9.
Table 9: amplification system
| Reagent(s) | Volume (20 mu L) |
| 2×Taq Plus Master Mix II(dye plus) | 10 |
| Primer F (10. Mu. Mol/. Mu.L) | 2 |
| Primer R (10. Mu. Mol/. Mu.L) | 2 |
| DNA template | 1 |
| ddH 2 O | 5 |
The PCR amplification procedure was: the denaturation temperature is 94 ℃ and the denaturation is carried out for 2min; then 15 cycles were performed in which the DNA was denatured at 98℃for 10s, renatured at 67℃at a decrease of 0.7℃per cycle for 30s, and extended at 68℃at a rate of 1 kb/min; further 25 cycles were performed in which the DNA was denatured at 98℃for 30s, renatured at 57℃for 30s, and extended at 68℃at a rate of 1 kb/min; maintaining at 68deg.C for 10min, and cooling to 4deg.C.
The products were subjected to gel electrophoresis after amplification, wherein the electrophoresis results of the CD34-L-GTF/CD34-L-GTR primer amplification products are shown in FIG. 8, and the electrophoresis results of the CD34-R-GTF/CD34-R-GTR primer amplification products are shown in FIG. 9, and the results indicate that F1 generation mice are positive for PCR in laboratory numbers 1E3Z3-002, 1E3Z3-003, 1E3Z3-005, 1E3Z3-007, 1E3Z3-014, 1E3Z3-015, 1E3Z3-025, 1E3Z3-032, 1E3Z3-036, 1E3Z3-040, 1E3Z 3-041E 3Z3-042, 1E3Z3-055, 1E3Z3-058, 1E3Z3-064, 1E3Z3-068, 1E3Z3-069, 1E3Z3-075 and 1E3Z 3-Z1 were positive for PCR in the first generation mice.
3. Southern blot detection of F1 generation positive mice
Extracting part of F1 generation mouse rat tail DNA identified as positive by genotype PCR, and carrying out Southern blot detection, wherein BglII and StuI are used as Southern blot enzyme cutting sites, cre Probe and 3'Probe are used as probes, and 3' Probe is used for detecting whether correct recombination occurs, and if so, two bands of wild type and mutant type can occur; if not properly recombined, only wild type bands will appear; the Cre Probe is used for detecting whether random insertion is contained or not, and if correct recombination occurs, only a target strip appears; if random insertion is present, multiple stripes will occur.
The Cre Probe (5') Probe sequence is shown below:
TGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATG(SEQ ID NO.19)。
the 3' probe sequence is shown below:
ACTTGGGATGGGCAGGGAATGAATCATTTCCCTAATGATGCAAAGGAAAAGGCCTCCTGGCTTAACTGGGAAGTCTGCTGCTTCCTGGAGTGGAAAATAAAAGCAAGCCTCTTAGAAAAGTGGTTATCTCTAGGTAGTTTGCTCCATTGCTTAAGAAAATGTGTAAATGGTTCTTTGGGTAAGAAATCTCAAAATGTTAGACAAGTTCTCAATATTCAAGGGCAGGCAGGAAGCAGGCTGGCTAAATGGGAAGTAGAGTCTTATATTGGAGTGGGTCAAGAGATGAAGGCATAAGGACTGCAGAATCTAAGGTAAAGACAAAAGGGTGACGCTCTTGCAGAGTTGGAAGCTTTAAAGGAGATTTCTTATAGAATAGCTTCATTAGCACAGTATTAACTAACATTTCAAGGTGTGCTACTGTCTCTTCTGGCTACCCTGAAATTTCACTCCATGCTTTCAAATCTCTGGTAAAAATTGCCCACGCATGGGAG(SEQ ID NO.20)。
the experimental results are shown in FIG. 10, wherein the Southern blot detection F1 generation mice with experimental numbers 1E3Z3-002, 1E3Z3-003, 1E3Z3-005 and 1E3Z3-007 are correctly recombined, randomly inserted and sequenced correctly, and are F1 generation randomly inserted positive mice; the test numbers 1E3Z3-014 and 1E3Z3-025 had correct recombination and no random insertion, and were Southern blot tested positive mice, i.e., target CD34 transgenic mode mice.
Example 6 tracking of CD34 Positive cell division differentiation Using CD34 transgenic Pattern mice
The cell division and differentiation conditions of the target CD34 transgenic mode mice positive in both the PCR verification and the Southern blot verification are tracked. The specific test method is as follows: CD34 CrePGR-tdTomato mice (i.e., CD34 transgenic mode mice described above) were taken at 7-8 weeks of resting stage and injected continuously with 5d RU486 (Sigma, in corn oil, 10 mg/ml). Skin samples were harvested on the sixth day and stained in sections after frozen embedding.
The staining procedure for frozen embedded sections was as follows: firstly, soaking skin tissues in 4% paraformaldehyde for 8 hours, then sequentially soaking and washing the skin tissues with 1X PBS for 8 hours, dehydrating the skin tissues with 30% sucrose for 8 hours, and then embedding the skin tissues in OCT (optical coherence tomography), and freezing the skin tissues to obtain sections with the thickness of 10 mu m; the sections were immersed in 1 XPBS for 10min, stained with DAPI (1 mg/mL) for 15min at room temperature, then washed with 1 XPBS for 10min, then blocked with anti-fluorescence quenchers, and finally scanned with a laser confocal microscope (Zeiss, LSN 880).
The results of the experiment are shown in FIG. 11, where blue is DAPI, the labeled nuclei, red is CD34+ cells, expressed with tdTomato, merge is a picture after pooling, from which it can be seen that in the CD34 transgenic mode mice can track CD34 positive cells, indicating that the CD34 transgenic mice were constructed successfully.
Further, a target CD34 transgenic mode mouse (CD 34 CrePGR-tdTomato) was crossed with mT/mG (JAX mic, no. 007576) mice to obtain CD34 CrePGR-tdTomato: mT/mG mice (abbreviated as CD34 mTMG mice) and follow CD34+ cell division differentiation.
The results are shown in FIG. 12, where DAPI is blue and the nuclei are labeled; green is the labeling of cell membranes, tracked cd34+ cells and their progeny; in red is a labeled cell membrane, mttdmamato represents membrane tdTomato; merge is the overlap of three colors. Fluorescence results showed that the construction of CD34 transgenic mice was successful, and their progeny CD34 mTMG mice were able to successfully track CD34+ cells and their progeny.
In summary, the invention provides a construction method and application of a CD34 transgenic mode mouse, wherein in the invention, firstly, a sgRNA is designed to construct a Cas9/sgRNA plasmid, then a CrePR1-P2A-tdTomato-WPRE-pA is knocked in at a fixed point at the initial codon site of a CD34 gene to construct a recombinant targeting vector, the Cas9/sgRNA plasmid and the recombinant targeting vector are injected into fertilized eggs of the mouse to develop so as to obtain an F0 generation mouse, then the F0 generation mouse with the genotype identified as positive is mated with a wild type mouse to obtain a positive F1 generation mouse, and the F1 generation positive mouse which is correctly recombined and is not randomly inserted is identified as a CD34 transgenic mouse model. After the CD34 transgenic mouse model is mated with other related system mice, the obtained offspring mice can realize the functions of specifically tracking the change of CD34 positive cells, over-expressing or knocking out target genes in the CD34 positive cells, and the like, and have important significance for the development of the transgenic mouse model.
While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (3)
1. A method for constructing a CD34 transgenic mode mouse, comprising the steps of:
s1, constructing a CRISPR/Cas9 system expression vector, wherein the CRISPR/Cas9 system expression vector consists of sgRNA with a nucleotide sequence shown as SEQ ID NO.4 or SEQ ID NO.5 and a skeleton vector pCS;
s2, constructing a recombinant targeting vector, wherein the recombinant targeting vector comprises a homologous arm at the 5 'end of a mouse CD34 gene, the mouse CD34 gene, a CrePR1-P2A-tdTomato-WPRE-pA sequence and a homologous arm at the 3' end of the mouse CD34 gene, and the CrePR1-P2A-tdTomato-WPRE-pA sequence is shown as SEQ ID NO. 14; the knockin site of the CrePR1-P2A-tdTomato-WPRE-pA sequence is between the start codon of the mouse CD34 gene and the exon1, and the skeleton carrier of the recombinant targeting carrier is a PUC19 carrier;
s3, introducing the CRISPR/Cas9 system expression vector and the recombinant targeting vector into fertilized eggs of C57BL/6N mice, screening F0-generation positive mice, hybridizing the F0-generation positive mice with wild C57BL/6N mice, and screening F1-generation recombinant positive mice to obtain the CD34 transgenic mode mice.
2. The method of claim 1, wherein the method of introducing comprises microinjection.
3. Use of the construction method according to claim 1 or 2 for obtaining mice with a specific follow-up of CD34 positive cell function.
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108103098A (en) * | 2017-12-14 | 2018-06-01 | 华南理工大学 | A kind of compound sensitization of skin evaluating in vitro cell model and its construction method |
| CN111500628A (en) * | 2020-04-15 | 2020-08-07 | 徐州医科大学 | Construction method and application of CD8 site-directed gene knock-in 2A-CreERT2-Wpre-pA mouse model |
| WO2022147347A1 (en) * | 2020-12-31 | 2022-07-07 | Vor Biopharma Inc. | Compositions and methods for cd34 gene modification |
| CN113897369A (en) * | 2021-09-07 | 2022-01-07 | 佛山市第一人民医院（中山大学附属佛山医院） | Construction and application of KRT10 site-specific gene knock-in P2A-CrePR1-T2A-tdTomato mouse model |
Non-Patent Citations (1)
| Title |
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| CRISPR/Cas9介导的CD34报告基因293AD细胞系的构建;吴福仁等;基因组学与应用生物学;第35卷(第3期);第472-478页 * |
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