CN113412820A - Construction and application of multi-organ cell gene mutation detection gene editing mouse model - Google Patents

Construction and application of multi-organ cell gene mutation detection gene editing mouse model Download PDF

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CN113412820A
CN113412820A CN202110690906.0A CN202110690906A CN113412820A CN 113412820 A CN113412820 A CN 113412820A CN 202110690906 A CN202110690906 A CN 202110690906A CN 113412820 A CN113412820 A CN 113412820A
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李海山
谢文平
沈国林
宋乃宁
赵潺
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Chinese Academy of Inspection and Quarantine CAIQ
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Abstract

The invention provides a mouse model edited by a CD59 gene, which is a mouse with a fixed-point knock-in CD 59-Enhanced Green Fluorescent Protein (EGFP) expression sequence segment. The invention also provides a method for constructing a mouse model edited by the CD59-EGFP gene, which is a method for knocking in a CD59-EGFP gene sequence at the site of mouse gene ROSA26 by using a CRISPR-Cas9 gene editing technology and comprises the following steps: designing and obtaining guide RNA, and preparing a guide RNA and Cas9 protein mixture; constructing a CD59-EGFP targeting vector; microinjection and F0 generation mice, F1 generation mice; in the CD59-EGFP targeting vector, the nucleotide sequence of CD59 is shown as SEQ ID NO.1, and the nucleotide sequence of EGFP is shown as SEQ ID NO. 2. The gene editing mouse model of the invention responds to mutagenic chemicals, separates liver cells and germ cells, can display CD59 cell membrane positioning through green fluorescence under a fluorescence microscope, and can be used for multi-organ cell mutagenicity detection of chemicals.

Description

Construction and application of multi-organ cell gene mutation detection gene editing mouse model
Technical Field
The invention relates to a multi-organ cell gene mutation detection gene editing mouse model, a construction method and application thereof, and belongs to the field of biochemistry.
Background
CD59 is a membrane regulatory protein with a molecular weight of 18-20 kDa. CD59 consists of 103 amino acid residues, contains a single N-terminal glycosylation site, and is anchored at the C-terminus to the cell surface by glycated phosphatidylinositol. CD59 is widely distributed and has been shown to be expressed on the skin, liver, kidney, pancreas, lung, salivary glands, nervous system, placenta, and various blood cells (erythrocytes, lymphocytes, neutrophils, and platelets) and sperm. The erythrocyte expression defect in anemia diseases such as paroxysmal nocturnal hemoglobinuria is commonly used as a biomarker of the diseases clinically. Studies have suggested that mutagenic chemicals may lead to reduced expression of CD59 on the surface of peripheral blood cells.
The human CD59 gene is located in the short arm of chromosome 11 and has homology with mouse CD 59. Therefore, a CD59 gene editing mouse introduced with fluorescent protein fusion expression is constructed, the fluorescence intensity on the surface of a cell membrane is directly observed under a fluorescence microscope, the gene mutation of various organ cells is conveniently and quickly reflected, and the method has advantages and application prospects.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a gene editing mouse model for expressing fluorescent protein fused CD59, which is applied to the detection of gene mutation of multi-organ cells.
Another object of the present invention is to provide a method for constructing the gene-editing mouse model.
The invention also aims to provide application of the gene editing mouse model in separating liver cells and germ cells and detecting gene mutation of different organs and cell types (somatic cells and germ cells).
In order to achieve the purpose, the invention adopts the following technical scheme:
first, a mouse model of CD59 gene editing is provided, which is a mouse that is spot-knocked-in with a fragment of the expression sequence of CD 59-Enhanced Green Fluorescent Protein (EGFP).
The mouse model for CD59 gene editing described in the present invention responds to mutagenic chemicals. The peripheral blood cells, the liver cells and the germ cells obtained by separation can be seen to have green fluorescence distributed in the cell membranes under a fluorescence microscope, and the green fluorescence intensity of the cell membranes is weakened when the mutant chemicals are given to mice.
The invention also provides a method for constructing a mouse model edited by the CD59 gene, which is a method for knocking in a CD59-EGFP gene sequence at the site of mouse gene ROSA26 by using a CRISPR-Cas9 gene editing technology and comprises the following steps: designing and obtaining guide RNA, and preparing a guide RNA and Cas9 protein mixture; constructing a CD59-EGFP targeting vector; microinjection was obtained with mice of the F0 generation and mice of the F1 generation.
In the preferable construction method of the invention, the CD59-EGFP targeting vector has a nucleotide sequence of CD59 shown in SEQ ID NO.1 and a nucleotide sequence of EGFP shown in SEQ ID NO. 2.
The most preferred construction method of the invention specifically comprises the following steps:
1) designing and obtaining CD59-EGFP guide RNA;
2) incubating the guide RNA obtained in 1) with a Cas9 protein to prepare a Cas 9/guide RNA mixture;
3) constructing a targeting vector, and constructing a CAG-Kozak-CD59-GS linker-EGFP-polyA homologous recombination vector by utilizing an In-Fusion cloning technology, wherein the nucleotide sequence of CD59 is shown as SEQ ID NO.1, and the nucleotide sequence of EGFP is shown as SEQ ID NO. 2;
4) co-injecting the Cas 9/guide RNA mixture obtained in the step 2) and the targeting vector obtained in the step 3) into pronucleus of fertilized eggs of a C57BL/6N mouse, transplanting the fertilized eggs after injection into a surrogate mother mouse for development, and screening out a midget mouse, namely an F0-generation mouse, through genotype identification after birth;
5) breeding the sexually mature mice in the F0-generation mice obtained in the step 4) with wild C57BL/6N mice to obtain F1-generation mice, and screening heterozygote mice which are transferred with the CD59-EGFP expression sequence segment from the F1-generation mice through genotype identification to obtain the CD59-EGFP transgenic mice capable of being bred by passage.
In a further preferred method of constructing a mouse model for CD59 gene editing according to the present invention, the guide RNA has the sequence: CTCCAGTCTTTCTAGAAGATGGG (SEQ ID NO. 3).
In a further preferred method of constructing a mouse model edited by the CD59 gene of the present invention, the step of genotyping described in step 4) and/or step 5) comprises: extracting mouse tail genome DNA by adopting PCR detection or sequencing verification, and performing PCR identification after targeting, wherein the primer sequences are as follows:
Figure BDA0003126128910000021
Figure BDA0003126128910000031
in a further preferred method of constructing a mouse model edited by CD59 gene of the present invention, in the PCR assay, if the DNA sample has a low purity or the PCR reaction time is desired to be shortened, then the PCR product with shorter fragments can be obtained by using the alternative PCR primers, wherein the sequences of the alternative PCR primers are as follows:
primer name Primer sequences
F4 5’-TCAAGATCCGCCACAACATCG-3’(SEQ ID NO.10)
R4 5’-CTTTATTAGCCAGAAGTCAGATGC-3’(SEQ ID NO.11)
。
In a further preferred method for constructing a CD59 gene editing mouse model according to the present invention, the genotyping described in step 5) is further verified by Southern blot, comprising extracting genomic DNA from mouse tail, digesting with restriction enzymes BamHI and BstEII, and selecting DNA probe sequences as follows:
5' Probe primer sequence:
primer name Primer sequences
Forward 5’-AAACGTGGAGTAGGCAATACCCAGG-3’(SEQ ID NO.12)
Reverse 5’-AAAGAAGGGTCACCTCAGTCTCCCT-3’(SEQ ID NO.13)
3' Probe primer sequence:
primer name Primer sequences
Forward 5’-TTCTGGGCAGGCTTAAAGGCTAAC-3’(SEQ ID NO.14)
Reverse 5’-AGGAGCGGGAGAAATGGATATGAAG-3’(SEQ ID NO.15)
。
The invention also provides application of the CD59-EGFP gene editing mouse model in rapid screening of mutagenic chemicals. In the application, peripheral blood cells of the mouse model are extracted, and liver or male germ cells of the mouse model edited by the CD59-EGFP gene are separated for detecting gene mutation of multi-organ cells.
The invention has the beneficial effects that: the bred gene-edited mouse widely expresses CD59-EGFP, and the green fluorescence intensity of peripheral red blood cells, isolated liver cells or the surface of a male germ cell membrane is weakened after the mouse is exposed to a mutagenic chemical. If equipped with flow cytometers, cryomicrotomes, and high content screening systems, the method of the invention can achieve rapid, high throughput, automated scanning and image analysis. Therefore, the gene editing mouse model can be used for rapidly detecting whether chemicals such as industrial raw material chemicals, micromolecule chemicals, pesticides and the like have mutagenicity, and has wide application prospect.
Drawings
FIG. 1 is a schematic diagram of the overall strategy for gene editing in the example of the present invention.
FIG. 2 is a schematic view of a targeting vector in an embodiment of the present invention.
FIG. 3 is a graph showing the result of Southern blot analysis of a target mouse in the example of the present invention.
FIG. 4 is a graph showing the statistics of fluorescence intensities of cells of different organs of mice administered with mutagenic chemicals in the experimental examples of the present invention.
Detailed Description
The invention is further illustrated by the following examples in conjunction with the accompanying drawings.
The invention establishes a transgenic mouse model expressing CD59-EGFP and has response to mutagenic chemicals.
Examples
The technical solution of the present invention is further explained with reference to the accompanying drawings.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail. The following examples are merely for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
In the following examples, reagents and biomaterials used were commercially available unless otherwise specified. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. In the examples, the molecular biology experimental methods not specifically described are described in molecular cloning, a manual (third edition) of experimental guidelines for molecular cloning (scientific Press, 2016, Sammbrook, Lassel), and the cell culture and experimental methods are described in culture cytology and cell culture techniques (Shanghai scientific technology Press, 2004, Zhang Nature).
The CD59-EGFP gene sequence is knocked into a mouse ROSA26 site by using a CRISPR-Cas9 gene editing technology, so that the over-expression of CD59-EGFP can be realized, and a mouse model for widely expressing CD59-EGFP is established. The method comprises the following steps:
1. guide RNA design and off-target effect assessment
Design of guide RNA at ROSA26 site of C57BL/6N mouse, and evaluation of off-target effect of candidate guide RNA. The sequence of the guide RNA obtained by screening is as follows: CTCCAGTCTTTCTAGAAGATGGG (SEQ ID NO. 3).
2. The resulting guide RNA of 1 was incubated with Cas9 protein to prepare a Cas 9/guide RNA mixture.
3. Gene editing strategy and targeting vector construction
The gene editing strategy is shown In figure 1, and In order to realize the gene editing purpose, a CAG-Kozak-CD59-GS linker-EGFP-polyA homologous recombination vector is constructed by utilizing an In-Fusion cloning technology as shown In figure 2. The nucleotide sequences of the CD59 and EGFP gene fragment are respectively shown as SEQ ID NO.1 and SEQ ID NO.2,
4. microinjection was performed with F0-generation mice
Co-injecting the targeting vector and the Cas 9/guide RNA mixture into pronuclei of fertilized eggs of a C57BL/6N mouse, transplanting the treated fertilized eggs into a surrogate mother mouse for development, and screening out a midget mouse, namely an F0-generation mouse, through genotype identification after birth.
Obtained from F1 mouse
And (3) breeding the sexually mature F0-generation mouse and a wild C57BL/6N mouse to obtain an F1-generation mouse, and screening a heterozygote mouse which is transformed into the CD59-EGFP fragment from the F1-generation mouse after PCR and Southern blot verification to finish the CD59-EGFP transgenic mouse capable of being bred by passage.
PCR detection and sequencing verification
The genotype identification in step 4 and the PCR verification in step 5 can be carried out in the following manner: extracting tail genome DNA of a mouse after pregnancy or a mouse generation F1, performing PCR identification after target hitting, respectively using primers F1/R1 and F2/R2 to amplify to generate PCR products to verify effective insertion, and using F3/R3 to sequence and compare the PCR products.
PCR primer sequences:
primer name Primer sequences
F1 5’-TACGCCACAGGGAGTCCAAGAATG-3’(SEQ ID NO.4)
R1 5’-GATGGGGAGAGTGAAGCAGAACG-3’(SEQ ID NO.5)
F2 5’-CTGCTGTCCATTCCTTATTCCATAG-3’(SEQ ID NO.6)
R2 5’-CTGGAAATCAGGCTGCAAATCTC-3’(SEQ ID NO.7)
F3 5’-CACTTGCTCTCCCAAAGTCGCTC-3’(SEQ ID NO.8)
R3 5’-ATACTCCGAGGCGGATCACAA-3’(SEQ ID NO.9)
And (3) PCR reaction system:
reaction assembly Volume of
Mouse tail genomic DNA 2μL
Forward primer (10. mu. mol/L) 2μL
Reverse primer (10. mu. mol/L) 2μL
dNTPs(2.5mmol/L) 6μL
5 XLongAmp Taq reaction solution 10μL
LongAmp Taq DNA polymerase 2μL
ddH2o 26μL
Total up to 50μL
And (3) PCR reaction conditions:
Figure BDA0003126128910000061
in this example, 3 positive F1 mice were obtained by PCR and sequencing verification, No. 10, No. 16, and No. 17.
If the DNA sample is less pure or if it is desired to shorten the PCR reaction time, then shorter fragment PCR products can be obtained using alternative PCR primers.
Alternative PCR primer sequences:
primer name Primer sequences
F4 5’-TCAAGATCCGCCACAACATCG-3’(SEQ ID NO.10)
R4 5’-CTTTATTAGCCAGAAGTCAGATGC-3’(SEQ ID NO.11)
Alternative PCR reaction system:
Figure BDA0003126128910000062
Figure BDA0003126128910000071
alternative PCR reaction conditions:
Figure BDA0003126128910000072
southern blot assay
Extracting the genome DNA of the double-arm homologous recombination positive mouse tail in the step 5 through PCR and sequencing verification, carrying out enzyme digestion by restriction enzymes BamHI and BstEII, and selecting a DNA probe. As shown in FIG. 3, the results show that the corresponding genome fragments of the three positive F1 mouse DNA fragments after digestion can be hybridized with the probe, and the size of the product is in accordance with the expectation.
The 5' probe primer sequences were as follows:
primer name Primer sequences
Forward 5’-AAACGTGGAGTAGGCAATACCCAGG-3’(SEQ ID NO.12)
Reverse 5’-AAAGAAGGGTCACCTCAGTCTCCCT-3’(SEQ ID NO.13)
The 3' probe primer sequences are as follows:
primer name Primer sequences
Forward 5’-TTCTGGGCAGGCTTAAAGGCTAAC-3’(SEQ ID NO.14)
Reverse 5’-AGGAGCGGGAGAAATGGATATGAAG-3’(SEQ ID NO.15)
Examples of the experiments
The CD59-EGFP transgenic mouse obtained in the embodiment is taken as an experimental animal to carry out a rapid screening experiment of mutagenic chemicals, and the specific steps are as follows:
male mice of 1.6-7 weeks of age were given the following chemicals:
ethyl methanesulfonate (EMS, CAS number: 759-73-9), gavage 10, 30, 90mg/kg/d orally for 3 consecutive days, using 3 mice per dose;
② N-ethyl-N-nitrosourea (ENU, CAS number: 759-73-9), gavage 10, 20, 40mg/kg/d by mouth, continuously 3 days, using 3 mice per dose;
③ Olive oil (OO, CAS number: 8001-25-0), gavage 10mL/kg/d orally for 3 consecutive days, using 3 mice;
after the test object is administered for 24 hours for the last time, tail venous blood is taken to prepare a blood smear which is observed under a fluorescence microscope.
2. Separating mouse liver cells by a two-step perfusion method, separating mouse testicular germ cells by a discontinuous gradient centrifugation method, and preparing a cell smear; the cell localization and fluorescence intensity of the green fluorescence were observed under a fluorescence microscope.
As shown in FIG. 4, compared with the green fluorescence intensity of peripheral blood cells, isolated hepatocytes and male germ cell membranes of mice in the negative control OO group, the mutating chemicals EMS and ENU all cause the reduction of the green fluorescence intensity of the membranes and show a dose-dependent decrease. The gene editing mouse model can be used for rapidly detecting whether chemicals such as industrial raw material chemicals, small molecular chemical drugs, pesticides and the like have mutagenicity.
Unless otherwise defined, terms used in the description of the present invention are terms well known in the related art. Standard chemical symbols and abbreviations may be used interchangeably with their full names.
Unless otherwise indicated, all techniques and methods used herein, which are not explicitly or implicitly set forth, are intended to be commonly used in the art and may be performed according to techniques and methods well known in the art. The use of the kit is according to the instructions provided by the manufacturer or supplier.
Sequence listing
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ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctccagtctt tctagaagat ggg 23
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tacgccacag ggagtccaag aatg 24
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gatggggaga gtgaagcaga acg 23
<210> 6
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ctgctgtcca ttccttattc catag 25
<210> 7
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ctggaaatca ggctgcaaat ctc 23
<210> 8
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cacttgctct cccaaagtcg ctc 23
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atactccgag gcggatcaca a 21
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tcaagatccg ccacaacatc g 21
<210> 11
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ctttattagc cagaagtcag atgc 24
<210> 12
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
aaacgtggag taggcaatac ccagg 25
<210> 13
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
aaagaagggt cacctcagtc tccct 25
<210> 14
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ttctgggcag gcttaaaggc taac 24
<210> 15
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
aggagcggga gaaatggata tgaag 25

Claims (9)

1. A mouse model of CD59 gene editing is a mouse of a fixed point knock-in CD 59-Enhanced Green Fluorescent Protein (EGFP) expression sequence segment.
2. A method for constructing a mouse model edited by a CD59-EGFP gene is a method for knocking in a CD59-EGFP gene sequence at the site of a mouse gene ROSA26 by using a CRISPR-Cas9 gene editing technology, and comprises the following steps: designing and obtaining guide RNA, and preparing a guide RNA and Cas9 protein mixture; constructing a CD59-EGFP targeting vector; microinjection and F0 generation mice, F1 generation mice; in the CD59-EGFP targeting vector, the nucleotide sequence of CD59 is shown as SEQ ID NO.1, and the nucleotide sequence of EGFP is shown as SEQ ID NO. 2.
3. The method for constructing a mouse model edited by a CD59-EGFP gene according to claim 2, which comprises the following steps:
1) designing and obtaining CD59-EGFP guide RNA;
2) incubating the guide RNA obtained in 1) with a Cas9 protein to prepare a Cas 9/guide RNA mixture;
3) constructing a targeting vector, and constructing a CAG-Kozak-CD59-GS linker-EGFP-polyA homologous recombination vector by utilizing an In-Fusion cloning technology, wherein the nucleotide sequence of CD59 is shown as SEQ ID NO.1, and the nucleotide sequence of EGFP is shown as SEQ ID NO. 2;
4) co-injecting the Cas 9/guide RNA mixture obtained in the step 2) and the targeting vector obtained in the step 3) into pronucleus of fertilized eggs of a C57BL/6N mouse, transplanting the fertilized eggs after injection into a surrogate mother mouse for development, and screening out a midget mouse, namely an F0-generation mouse, through genotype identification after birth;
5) breeding the sexually mature mice in the F0-generation mice obtained in the step 4) with wild C57BL/6N mice to obtain F1-generation mice, and screening heterozygote mice which are transferred with the CD59-EGFP expression sequence segment from the F1-generation mice through genotype identification to obtain the CD59-EGFP transgenic mice capable of being bred by passage.
4. The method of constructing a mouse model of CD59-EGFP gene editing as claimed in any of claims 2 or 3, wherein the sequence of the guide RNA is as shown in SEQ ID No. 3.
5. The method of constructing a mouse model of CD59-EGFP gene editing according to claim 3, wherein the step of genotyping in step 4) and/or step 5) comprises: extracting mouse tail genome DNA by adopting PCR detection or sequencing verification, and performing PCR identification after targeting, wherein the primer sequences are as follows:
Figure FDA0003126128900000011
Figure FDA0003126128900000021
6. the method of claim 5 for constructing a mouse model of CD59-EGFP gene editing, wherein the PCR assay employs PCR primers having the following sequences to obtain shorter fragment PCR products:
primer name Primer sequences F4 5’-TCAAGATCCGCCACAACATCG-3’(SEQ ID NO.10) R4 5’-CTTTATTAGCCAGAAGTCAGATGC-3’(SEQ ID NO.11)
。
7. The method of claim 5 for constructing a mouse model of CD59-EGFP gene editing, wherein the genotyping in step 5) is further verified by Southern blot comprising extracting genomic DNA from mouse tail, cleaving with the restriction enzymes BamHI and BstEII, and selecting DNA probe sequences as follows:
5' Probe primer sequence:
primer name Primer sequences Forward 5’-AAACGTGGAGTAGGCAATACCCAGG-3’(SEQ ID NO.12) Reverse 5’-AAAGAAGGGTCACCTCAGTCTCCCT-3’(SEQ ID NO.13)
3' Probe primer sequence:
primer name Primer sequences Forward 5’-TTCTGGGCAGGCTTAAAGGCTAAC-3’(SEQ ID NO.14) Reverse 5’-AGGAGCGGGAGAAATGGATATGAAG-3’(SEQ ID NO.15)
8. Use of the CD59-EGFP gene-edited mouse model constructed according to the method of claim 2 for the detection of mutagenic chemicals.
9. Use according to claim 8, characterized in that: peripheral blood of the mouse model is collected, and liver cells and/or male germ cells of the mouse model are separated and then used for detection of mutagenic chemicals.
CN202110690906.0A 2021-06-22 2021-06-22 Construction and application of multi-organ cell gene mutation detection gene editing mouse model Pending CN113412820A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110771573A (en) * 2019-11-15 2020-02-11 西安医学院 Mouse animal model with knocked-in PirB gene and construction method thereof
CN111019971A (en) * 2019-12-19 2020-04-17 上海同科生物科技有限公司 Construction method of mouse model for conditionally overexpressing HPV E6 gene at ROSA26 site
CN111718932A (en) * 2020-06-08 2020-09-29 中国人民解放军军事科学院军事医学研究院 Preparation method and application of novel gene editing animal bioreactor

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110771573A (en) * 2019-11-15 2020-02-11 西安医学院 Mouse animal model with knocked-in PirB gene and construction method thereof
CN111019971A (en) * 2019-12-19 2020-04-17 上海同科生物科技有限公司 Construction method of mouse model for conditionally overexpressing HPV E6 gene at ROSA26 site
CN111718932A (en) * 2020-06-08 2020-09-29 中国人民解放军军事科学院军事医学研究院 Preparation method and application of novel gene editing animal bioreactor

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GENBANK: "GenBank: MN832872.1", 《GENBANK: MN832872.1》 *
NCBI: "NCBI Reference Sequence: NM_000611.6", 《NCBI REFERENCE SEQUENCE: NM_000611.6》 *
ZHIYING JI等: "Applying the Erythrocyte Pig-a Assay Concept to Rat Epididymal Sperm for GermCellMutagenicity", 《ENVIRONMENTAL AND MOLECULAR MUTAGENESIS》 *
刘香梅 等: "大鼠Pig-α基因突变试验中N-乙基-N-亚硝基脲和环磷酰胺量-效关系的优化", 《中国比较医学杂志》 *
肖红卫 等: "分子技术对表达GFP转基因胚胎的再检测", 《湖北农业科学》 *
郑丹丹 等: "Pig-a 基因突变试验研究进展", 《中国比较医学杂志》 *

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