CN113584030A - Construction method of CNN3 gene knockout mouse model based on Cre-FloxP system - Google Patents
Construction method of CNN3 gene knockout mouse model based on Cre-FloxP system Download PDFInfo
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Abstract
The invention discloses a construction method of a CNN3 gene knockout mouse model based on a Cre-FloxP system, which comprises the following steps: FloxP gene segments are respectively inserted into two sides of a second exon of a CNN3 gene to construct a Cas9/gRNA target spot and construct a homologous recombination template, the homologous recombination template and donor DNA are injected into fertilized eggs of mice together in a micro-injection mode to obtain a founded Cnn3-FloxP mouse, and the founded mouse and the female mouse are copulated and selfed to obtain a stably inherited homozygote CNN3fl/flA mouse; subjecting said homozygote CNN3fl/flMating the mice with tissue-specifically expressed Cre miceSelfing, and screening out the genotype as Cre by gene identification+/‑/CNN3fl/flA mouse. The invention relates to the technical field of gene design, can efficiently construct a mouse with a specific tissue or a specific cell CNN3 gene knockout, and greatly improves the construction efficiency and the construction success rate of a selective knockout CNN3 mouse model.
Description
Technical Field
The invention relates to the technical field of genes, in particular to a construction method of a CNN3 gene knockout mouse model based on a Cre-FloxP system.
Background
The Calponin protein is a binding protein for calcium/calmodulin and actin, and has 3 subtypes: calponin-1(basic calponin), calponin-2 (neutralcopin), and calponin-3(acid calponin). calponin-3 is the only subset of the calponin family that is distributed in the central nervous system and is primarily concentrated in astrocytes and neurons in the brain.
The regulatory gene CNN3 of calponin-3 protein is located on chromosome 1p 21-22. CNN3 was initially found to regulate neural tube morphogenesis during embryonic development. Due to defects in central nervous system development, systemic knockdown of CNN3 in mice resulted in embryonic and neonatal death. Subsequent studies provide evidence of the function of CNN3 beyond the field of neural growth development. Calponin 3(CNN3) is an F-actin binding protein that regulates actin cytoskeletal rearrangement, and CNN3 plays a role in cell differentiation, proliferation, and migration by participating in stress fiber formation or cytoskeletal remodeling during cell fusion of trophoblast and myoblast cells, and cell movement and contraction of dermal fibroblasts.
In the research of the participation of CNN3 in the development of cancer, CNN3 is found to be related to colorectal cancer lymph node metastasis and peritoneal metastasis and to be involved in lymph node metastasis of colon cancer and chemotherapy-resistant reaction. In the gastric cancer study, the CNN3 expression is remarkably increased in the highly aggressive cancer cell line compared with the less aggressive or non-aggressive cancer cell line, and the invasion capacity of the gastric cancer cells is inhibited by the consumption of the CNN3 protein; highly invasive MKN-28 gastric cancer cells are more resistant to doxorubicin than non-invasive MKN-45 cells, the knockdown of CNN3 expression in MKN-28 cells makes them re-sensitive to doxorubicin therapy, CNN3 plays a key role in gastric cancer cell invasiveness and doxorubicin resistance. Recent studies have shown that CNN3 can become an oncogene by affecting the expression of RPLP1 mRNA, as a potential target for blocking cervical cancer metastasis.
The gene chip scanning is carried out on the temporal lobe tissue of a drug-resistant epileptic, so that the CNN3 gene is found to be remarkably up-regulated, and the calponin-3 protein is found to be abnormally expressed in the neurons and astrocytes of the brain tissue of the patient. The CNN3 gene silencing/over-expression lentivirus vector developed and constructed is injected into local brain areas of experimental mice by adopting a stereotaxic injection method, and the calponin-3 is found to influence the epilepsy susceptibility of the experimental mice, but the specific action link is unclear. Under the background that embryo death can occur in the systematic knockout of CNN3, in order to deeply research the mechanism of action of CNN3 in multidisciplinary and multi-field research, a selective knockout CNN3 mouse model is urgently needed to be constructed. The prior art method has the problem that the selective knockout CNN3 mouse model is difficult to efficiently construct.
Disclosure of Invention
The embodiment of the invention provides a method for constructing a CNN3 gene knockout mouse model based on a Cre-FloxP system, and aims to solve the problem that the efficiency of constructing a selective knockout CNN3 mouse model in the prior art is not high.
The embodiment of the invention provides a construction method of a CNN3 gene knockout mouse model based on a Cre-FloxP system, which comprises the following steps:
inserting FloxP gene fragments into two sides of a second exon of the CNN3 gene to obtain donor DNA;
constructing a Cas9/gRNA target point, wherein the Cas9/gRNA target point comprises a Cnn3-L1 gene segment and a Cnn3-R1 gene segment;
respectively carrying out in vitro transcription on the Cnn3-L1 fragment, the Cnn3-R1 fragment and the Cas9 endonuclease to construct a homologous recombination template so as to obtain corresponding mRNA and RNA;
injecting mRNA and RNA corresponding to the Cnn3-L1 fragment, the Cnn3-R1 fragment and the Cas9 endonuclease respectively into fertilized eggs of mice together with the donor DNA to obtain a founded Cnn3-FloxP mouse;
mating the pioneer Cnn3-FloxP mouse and female mouse for selfing to obtain stable genetic homozygote CNN3fl/flA mouse;
subjecting said homozygote CNN3fl/flMating the mouse with tissue-specific Cre mouse to obtain Cre+/-/CNN3fl /wtMice are selfed and genotype Cre is screened out by gene identification+/-/CNN3fl/flA mouse.
The embodiment of the invention provides a construction method of a CNN3 gene knockout mouse model based on a Cre-FloxP system. The method comprises the following steps: FloxP gene segments are respectively inserted into two sides of a second exon of a CNN3 gene to construct a Cas9/gRNA target spot and construct a homologous recombination template, the homologous recombination template and donor DNA are injected into fertilized eggs of mice together in a micro-injection mode to obtain a founded Cnn3-FloxP mouse, and the founded mouse and the female mouse are copulated and selfed to obtain a stably inherited homozygote CNN3fl/flA mouse; subjecting said homozygote CNN3fl/flThe mice are copulated with Cre mice expressed by tissue specificity, selfed and screened out the genotype as Cre through gene identification+/-/CNN3fl/flA mouse. By the method, the mouse with the specific tissue or the specific cell CNN3 knocked out can be efficiently constructed, and the construction efficiency and the construction success rate of the selective knock-out CNN3 mouse model are greatly improved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic flow chart of a method for constructing a CNN3 gene knockout mouse model based on a Cre-FloxP system according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a mouse pedigree of a CNN3 gene knockout mouse model based on a Cre-FloxP system according to an embodiment of the present invention;
FIG. 3 is another schematic diagram of a mouse pedigree of a CNN3 gene knockout mouse model based on Cre-FloxP system according to an embodiment of the present invention;
FIG. 4 is a schematic diagram showing the effect of the method for constructing a CNN3 gene knockout mouse model based on a Cre-FloxP system according to the embodiment of the present invention;
FIG. 5 is a schematic diagram showing the effect of the method for constructing a CNN3 gene knockout mouse model based on a Cre-FloxP system according to the embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Introduction of raw materials and equipment in the examples:
experimental mice, with a genetic background of C57BL/6J, were housed in the kunming institute SPF (Specific pathognomone free, pathogen free) environment;
PCR primers, purchased from Ongchow Bio Inc.;
PCR assay kit, purchased from Vazyme;
calponin3 Rabbit polyclonal antibody, available from abcam corporation under the cat number ab 151427;
the genetic tool mouse (C57BL/6 genetic background) for systemic expression or tissue-specific expression of Cre enzyme is provided by animal research institute of Chinese academy of sciences;
cas9/gRNA target efficiency detection kit, purchased from Beijing Weishanglide, having a product number of VK 007; .
It will be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
It is also to be understood that the terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the specification of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
It should be further understood that the term "and/or" as used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.
Referring to fig. 1, fig. 1 is a schematic flow chart of a method for constructing a CNN3 knockout mouse model based on a Cre-FloxP system according to an embodiment of the present invention, as shown in fig. 1, the method includes steps S110 to S160.
S110, FloxP gene fragments are inserted into both sides of the second exon of CNN3 gene to obtain donor DNA.
S120, constructing a Cas9/gRNA target point, wherein the Cas9/gRNA target point comprises a Cnn3-L1 gene segment and a Cnn3-R1 gene segment.
Designing and constructing a Cas9/gRNA target point, wherein the Cas9/gRNA target point comprises a Cnn3-L1 gene segment and a Cnn3-R1 gene segment, and in a more specific embodiment, the sequence of the Cnn3-L1 gene segment is CTTGCGTCATGCGTGACGGGG; the sequence of the Cnn3-R1 gene fragment is AGGCACAAGCCCACAGACAGG. Specifically, a Beijing Weishangride Cas9/gRNA target point efficiency detection kit can be adopted to detect the in vitro enzyme digestion activity of the Cas9/gRNA target points, a Cas9/gRNA gene segment with high in vitro enzyme digestion activity is selected for subsequent use, and Cas9/gRNA can be selected for transcription to obtain corresponding mRNA and RNA, and the mRNA and the RNA are subjected to microinjection to fertilized eggs; then embryo can be taken to detect the endogenous activity of the Cas9/gRNA target point, and the Cas9/gRNA target point with higher endogenous activity is selected for subsequent experiments according to the detection result of the endogenous activity of the Cas9/gRNA target point.
S130, respectively carrying out in vitro transcription on the Cnn3-L1 fragment, the Cnn3-R1 fragment and the Cas9 endonuclease to construct a homologous recombination template, and obtaining corresponding mRNA and RNA.
S140, injecting mRNA and RNA corresponding to the Cnn3-L1 fragment, the Cnn3-R1 fragment and the Cas9 endonuclease respectively into fertilized eggs of mice together with the donor DNA to obtain the founding Cnn3-FloxP mice.
Specifically, the Cas9 endonuclease is Cas9nickase, Cas9 endonuclease, Cnn3-L1 and Cnn3-R1 are subjected to in vitro transcription to obtain mRNA and RNA, then Donor DNA (Donor DNA) is added and is injected into fertilized eggs of a mouse (genetic background is C57BL/6J) in a micro mode, after the mouse is born for 2 weeks, the mouse tail is cut, mouse genome DNA is extracted, PCR (polymerase chain reaction) is carried out by using PCR primers to replicate the mouse DNA, the mouse DNA is sequenced by using a PCR identification kit to detect the genotype of the mouse, whether accurate insertion of FloxP at two sites is realized or not is detected by sequencing, and the initial Cnn3-FloxP mouse with knocked-in FloxP genes is efficiently obtained; wherein the gene sequence of Cnn3-L1 is CTTGCGTCATGCGTGACGGGG, and the gene sequence of Cnn3-R1 is AGGCACAAGCCCACAGACAGG.
Fig. 4 is a schematic diagram showing the effect of the method for constructing the CNN3 gene knockout mouse model based on the Cre-FloxP system provided in the embodiment of the present invention, and the genotype identification result of the CNN3-FloxP homozygous mouse is shown in fig. 4.
S150, mating the pioneer Cnn3-FloxP mouse and a female mouse for selfing to obtain a stable genetic homozygote CNN3fl/flA mouse; wherein the female mouse is C57BL/6J female mouse, specifically, the pioneer Cnn3-FloxP mouse is mated with the female mouse when the mouse reaches 6-8 weeks of age, and a stably inherited heterozygote CNN3 is obtainedfl/wtMouse, stably inherited heterozygote CNN3 obtainedfl/wtMice were selfed to obtain homozygous CNN3fl/flFIG. 2 is a mouse genealogical diagram of a CNN3 gene knockout mouse model based on Cre-FloxP system according to an embodiment of the present invention, and the CNN3 is homozygousfl/flThe procedure for obtaining the mice is shown in FIG. 2.
S160, carrying out CNN3 on the homozygotefl/flMating the mouse with tissue-specific Cre mouse to obtain Cre+/-/CNN3fl/wtMice are selfed, and the genotype is screened out as Cre through gene identification+/-/CNN3fl/flA mouse.
Mixing CNN3fl/flHybridizing the mouse with a tool mouse for expressing Cre enzyme specifically by the neuron cells to obtain Cre+/-/CNN3fl /wtMice F1 generation and F1 generation are self-bred, and can be screened by gene adherence to obtain Cre+/-/CNN3fl/flA mouse. FIG. 3 is another mouse genealogical diagram of CNN3 gene knockout mouse model based on Cre-FloxP system according to the present invention, Cre+/-/CNN3fl/flThe mouse acquisition process is shown in FIG. 3, in which the WT allele (WT allele) is 124bp and the MT allele (MT allele) is 158 bp. Specifically, mouse tail genomic DNA of a mouse after selfing of an F1-generation mouse can be extracted, genotype identification is respectively carried out, and Cre can be screened from the F1-generation mouse through the genotype identification+/-/CNN3fl/flThe specific genotype identification process of the mouse is the same as the process for identifying whether the mouse genotype realizes the accurate insertion of the FloxP at two sites. Wherein the gene sequence of F1 in the Cre analysis primer used in the genotype identification process is 5'-TCGATGCAACGAGTGATGAG-3', and the gene sequence of R1 is 5'-TCCATGAGTGAACGAACCTG-3'; the gene sequence of F2 in the analysis primer for the insertion condition of the L-terminal Floxp gene fragment is 5'-TGCCTTCACTCACAGTCT-3', and the gene sequence of R2 is 5'-CGTTCATTGTCCACTAAGC-3'; f in analysis primer of insertion condition of R-terminal Floxp gene fragment3 is 5'-GAAGGACGGCATCATATTG-3', and R3 is 5'-AGAATAACACAGTGGTAGGA-3', and the above analytical primers can be used to identify heterozygous knockout and homozygous knockout.
In one embodiment, as shown in fig. 1, step S170 is further included after step S160.
S170, detecting the Cre by adopting Westernblot+/-/CNN3fl/flThe protein Calponin-3 expression change coded by the mouse CNN3 gene is used for verifying the Cre+/-/CNN3fl/flWhether the mouse is a CNN3 gene knockout mouse.
Specifically, the Cre can be detected by Western blot+/-/CNN3fl/flThe expression change of the protein calcinin-3 coded by the CNN3 gene of the mouse can verify Cre+/-/CNN3fl/flWhether the mouse is a CNN3 gene knockout mouse or not, and particularly, the Cre screened by the genotype identification+/-/CNN3fl/flExtracting tissue proteins of a mouse and a wild-type mouse, wherein the tissue dissection, homogenization and cracking are carried out, Westernblot detection is carried out on an extracted protein sample according to a Calponin3 rabbit polyclonal antibody, fig. 5 is an effect schematic diagram of a construction method of a CNN3 gene knockout mouse model based on a Cre-FloxP system provided by the embodiment of the invention, KO in fig. 5 is a genotype-identified Cre +/-/CNN3fl/fl mouse, "WT" is a wild-type mouse, "Calponin-3" shows the expression condition of proteins coded by knockout genes CNN3 of the mouse, and "Tublin" is an internal reference protein. The detection result of Western blot detection shows that the CNN3 gene knockout mouse model is successfully constructed by the implementation method, and the constructed CNN3 gene knockout mouse model is an important animal model which can be used for later detection and research on physiological functions and pathological defects of the CNN3 gene.
In the method for constructing the CNN3 gene knockout mouse model based on the Cre-FloxP system, the method comprises the following steps: FloxP gene fragments are respectively inserted into two sides of a second exon of a CNN3 gene to construct a Cas9/gRNA target spot and construct a homologous recombination template, and the homologous recombination template and the donor DNA are injected into a germ cell of a mouse together to obtain a starting Cnn3FloxP mice, and mating with female mice for selfing to obtain stably inherited homozygote CNN3fl/flA mouse; subjecting said homozygote CNN3fl/flThe mice are copulated with Cre mice expressed by tissue specificity, selfed and screened out the genotype as Cre through gene identification+/-/CNN3fl/flA mouse. The invention relates to the technical field of gene design, can efficiently construct a mouse with a specific tissue or a specific cell CNN3 gene knockout, and greatly improves the construction efficiency and the construction success rate of a selective knockout CNN3 mouse model.
While the invention has been described with reference to specific embodiments, the invention is not limited thereto, and various equivalent modifications and substitutions can be easily made by those skilled in the art within the technical scope of the invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (5)
1. A construction method of a CNN3 gene knockout mouse model based on a Cre-FloxP system is characterized by comprising the following steps:
inserting FloxP gene fragments into two sides of a second exon of the CNN3 gene to obtain donor DNA;
constructing a Cas9/gRNA target point, wherein the Cas9/gRNA target point comprises a Cnn3-L1 gene segment and a Cnn3-R1 gene segment;
respectively carrying out in vitro transcription on the Cnn3-L1 fragment, the Cnn3-R1 fragment and the Cas9 endonuclease to construct a homologous recombination template so as to obtain corresponding mRNA and RNA;
injecting mRNA and RNA corresponding to the Cnn3-L1 fragment, the Cnn3-R1 fragment and the Cas9 endonuclease respectively into fertilized eggs of mice together with the donor DNA to obtain a founded Cnn3-FloxP mouse;
mating the pioneer Cnn3-FloxP mouse and female mouse for selfing to obtain stable genetic homozygote CNN3fl/flA mouse;
subjecting said homozygote CNN3fl/flMouse and tissue-specific expressed Cre miceMating mice to obtain Cre+/-/CNN3fl/wtMice are selfed and genotype Cre is screened out by gene identification+/-/CNN3fl/flA mouse.
2. The method for constructing a CNN3 gene knockout mouse model based on Cre-FloxP system of claim 1, wherein the sequence of the Cnn3-L1 gene fragment is CTTGCGTCATGCGTGACGGGG; the sequence of the Cnn3-R1 gene fragment is AGGCACAAGCCCACAGACAGG.
3. The method for constructing the CNN3 gene knockout mouse model based on the Cre-FloxP system of claim 1, wherein the female mouse is a C57BL/6J female mouse.
4. The method for constructing a CNN3 gene knockout mouse model based on Cre-FloxP system of claim 3, wherein the founding Cnn3-FloxP mouse is mated with the female mouse when it is 6-8 weeks old, and a heterozygote CNN3 with stable inheritance is obtainedfl/wtMouse, said heterozygote CNN3fl/wtInbreeding of mice to obtain stably inherited homozygote CNN3fl/flA mouse.
5. The method for constructing CNN3 gene knockout mouse model based on Cre-FloxP system of claim 1, wherein the genotype Cre is screened by gene identification+/-/CNN3fl/flAfter the mice, the method also comprises the following steps:
detecting the Cre by Western blot+/-/CNN3fl/flThe protein Calponin-3 expression change coded by the mouse CNN3 gene is used for verifying the Cre+/-/CNN3fl/flWhether the mouse is a CNN3 gene knockout mouse.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114223613A (en) * | 2021-11-30 | 2022-03-25 | 西安医学院 | Mouse model for hypercholesterolemia and atherosclerosis induced by AAV8-PCSK9 and construction method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104404036A (en) * | 2014-11-03 | 2015-03-11 | 赛业（苏州）生物科技有限公司 | Conditional gene knockout method based on CRISPR/Cas9 technology |
| CN107858373A (en) * | 2017-11-16 | 2018-03-30 | 山东省千佛山医院 | Endothelial cell conditionity knocks out the construction method of CCR5 genetic mouse models |
-
2021
- 2021-08-18 CN CN202110946373.8A patent/CN113584030A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104404036A (en) * | 2014-11-03 | 2015-03-11 | 赛业（苏州）生物科技有限公司 | Conditional gene knockout method based on CRISPR/Cas9 technology |
| CN107858373A (en) * | 2017-11-16 | 2018-03-30 | 山东省千佛山医院 | Endothelial cell conditionity knocks out the construction method of CCR5 genetic mouse models |
Non-Patent Citations (5)
| Title |
|---|
| ALEXANDRA FLEMMING等: "A conditional knockout mouse model reveals that calponin-3 is dispensable for early b cell development", 《PLOS ONE》 * |
| ALEXANDRA FLEMMING等: "A conditional knockout mouse model reveals that calponin-3 is dispensable for early b cell development", 《PLOS ONE》, vol. 10, no. 6, 5 June 2015 (2015-06-05), pages 0128385 * |
| GENBANK: "Mus musculus strain C57BL/6J chromosome 3, GRCm39", 《GENBANK》 * |
| GENBANK: "Mus musculus strain C57BL/6J chromosome 3, GRCm39", 《GENBANK》, 22 September 2020 (2020-09-22), pages 000069 * |
| KATARZYNA CIUBA等: "Calponin-3 is critical for coordinated contractility of actin stress fibers", 《SCIENTIFIC REPORTS》, vol. 8, no. 1, pages 17670 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114223613A (en) * | 2021-11-30 | 2022-03-25 | 西安医学院 | Mouse model for hypercholesterolemia and atherosclerosis induced by AAV8-PCSK9 and construction method |
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