CN114134216A - A method and application for rapid identification of MSC gene modification based on PCR - Google Patents
A method and application for rapid identification of MSC gene modification based on PCR Download PDFInfo
- Publication number
- CN114134216A CN114134216A CN202111295836.5A CN202111295836A CN114134216A CN 114134216 A CN114134216 A CN 114134216A CN 202111295836 A CN202111295836 A CN 202111295836A CN 114134216 A CN114134216 A CN 114134216A
- Authority
- CN
- China
- Prior art keywords
- msc
- pcr
- identification
- gene
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供了一种基于PCR快速鉴定MSC基因修饰的方法与应用。本发明提供的快速鉴定方法，只需要极少的细胞即可完成鉴定，在基因打靶结束后，最快仅需10天即可得到足够的细胞进行鉴定，大大节约了时间，使得靶向基因组改造在MSC中可以有效的实现。鉴定只需要极少的细胞也大大减少了MSC在体外扩增的时间，避免了因MSC体外扩增时间过长导致的功能下降。不仅如此，本发明筛选出来用于后续工作的阳性克隆，扩增时间短，传代次数较少，在干性维持、分化潜能等方面也具有一定的优势。除此之外，PCR鉴定过程操作简单、方便，对操作人员和仪器要求不高；耗时短，只需几个小时即可完成；可批量化操作，允许一次鉴定几十甚至上百个克隆，具有极高的应用前景。
The invention provides a method and application for rapidly identifying MSC gene modification based on PCR. The rapid identification method provided by the invention can complete the identification with only a few cells. After the end of gene targeting, enough cells can be obtained for identification in as little as 10 days, which greatly saves time and enables targeted genome modification. It can be effectively implemented in MSC. The identification requires only a few cells and also greatly reduces the time of MSC expansion in vitro, and avoids the functional decline caused by the long expansion time of MSC in vitro. Not only that, the positive clones screened out by the present invention for follow-up work have short expansion time and fewer passages, and also have certain advantages in stemness maintenance, differentiation potential and the like. In addition, the PCR identification process is simple and convenient, and does not require high operators and instruments; it takes only a few hours to complete; it can be operated in batches, allowing dozens or even hundreds of clones to be identified at one time , has a very high application prospect.
Description
Technical Field
The invention relates to the fields of genetic engineering and biomedicine, in particular to a method for rapidly identifying MSC gene modification based on PCR and application thereof.
Background
Mesenchymal Stem Cells (MSCs) are adult Stem Cells with a variety of differentiation and self-renewal capabilities. It is widely used in research related to regenerative medicine and disease treatment due to its low immunogenicity and immunoregulatory ability. However, adult MSCs also decline rapidly with age. In addition, various diseases can also significantly affect the function of MSCs. Therefore, before autologous MSC transplantation, the targeting genome editing technology is utilized to carry out targeting modification on the MSC, and the method has important significance.
After targeted editing of the genome, the edited cells need to be identified. The traditional identification method and process is to prepare single cell clone, then to expand to a certain cell number, and then to perform DNA extraction and related identification work. However, since the process takes a long time, and the function of the MSC is reduced due to the long time of in vitro amplification, the genome targeting modification and identification work of the MSC is limited by certain technology.
Therefore, the prior art has yet to be improved.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a method for rapidly identifying MSC gene modification based on PCR and application thereof, and aims to solve the problems that the identification process is long in time consumption and large in required cell amount after MSC gene modification in the prior art, and further the next screening and application are restricted.
The technical scheme of the invention is as follows:
in a first aspect, the present invention provides a method for rapidly identifying MSC gene modifications based on PCR, which comprises the steps of:
A) modifying the specific gene of the MSC by using a Cas9 gene targeting technology;
B) after gene targeting is finished, recovering for 3 days according to 80% cell density, then inoculating the cells into a 96-well plate according to the density of 0.8 cell per well, and culturing and maintaining by using an MSC conditioned medium;
C) after 7 days, the cells from each well were divided into two, one for continuous culture and the other for rapid PCR-based identification;
D) and (4) screening positive clones according to the result of the rapid identification, and carrying out the next analysis after amplification.
The method for rapidly identifying MSC gene modification based on PCR comprises the following specific steps:
1) cells were rapidly centrifuged at 5000rpm for 1min and resuspended in 15. mu.l of lysate;
2) keeping at 55 deg.C for 60 min;
3) keeping at 95 deg.C for 15 min;
4) quickly centrifuging at 12000rpm for 1min to obtain supernatant for subsequent PCR identification;
5) taking the supernatant, adding a specific primer aiming at the modified MSC specific gene, and carrying out PCR amplification;
6) the size of the PCR amplification product was identified and compared to the expected fragment size.
The method for rapidly identifying MSC gene modification based on PCR comprises the steps of dissolving the mixture into 10mM Tris-HCl with the pH value of 8.0, 2mM EDTA with the pH value of 8.0, 2% Tween 20 and 1mg/ml proteinase K.
The method for rapidly identifying the MSC gene modification based on the PCR comprises the steps of screening positive clones according to a rapid identification result, and carrying out modified gene expression quantity analysis, MSC marker analysis and MSC differentiation capacity identification after amplification.
In a second aspect, the present invention provides the use of a method for rapid identification of genetic modifications of MSCs based on PCR, wherein any of the methods described above is used for identification of genetic modifications of MSCs.
Has the advantages that: the invention provides a method for rapidly identifying MSC gene modification based on PCR and application thereof. The method for rapidly identifying based on PCR provided by the invention can complete identification only by a few cells, and can obtain enough cells for identification only by 10 days at the fastest speed after gene targeting is finished, so that the time is greatly saved, the progress of subsequent work is greatly promoted, and targeted genome modification can be effectively realized in MSC. Moreover, identification only needs few cells, so that the time of in vitro expansion of the MSC is greatly shortened, and the function reduction caused by overlong time of in vitro expansion of the MSC is avoided. Moreover, compared with the traditional technology, the method screens out positive clones for subsequent work, has short amplification time and less passage times, and also has certain advantages in the aspects of dryness maintenance, differentiation potential and the like. In addition, the PCR identification process is simple and convenient to operate, and has low requirements on operators and instruments; the time consumption is short, and the operation can be completed in a few hours; can be operated in batch, allows dozens or even hundreds of clones to be identified at one time, and has extremely high application prospect.
Drawings
FIG. 1 is a flowchart illustrating a method for rapidly identifying MSC gene modification based on PCR according to an embodiment of the present invention.
FIG. 2 is a schematic diagram of targeting vectors and targeting sites for the integration of the inflammation inhibitory factor IL-10 into the AASV1 genomic locus in an embodiment of the present invention.
FIG. 3 is a diagram illustrating the results of the PCR-based rapid identification method according to an embodiment of the present invention.
FIG. 4 is a diagram showing the results of conventional PCR identification after amplification in the example of the present invention.
FIG. 5 is an analysis of the expression level of IL-10 of the positive clone in the example of the present invention.
FIG. 6 is a logistic analysis of MSC markers for positive clones in the examples of the present invention.
FIG. 7 is a diagram showing the results of the differentiation ability assay of positive clones in the examples of the present invention.
Detailed Description
The invention provides a method for rapidly identifying MSC gene modification based on PCR and application thereof, and the invention is further described in detail below in order to make the purpose, technical scheme and effect of the invention clearer and more clear. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In a first aspect, embodiments of the present invention provide a method for rapidly identifying MSC gene modifications based on PCR, as shown in fig. 1, including the steps of:
s10, modifying the specific gene of the MSC by using a Cas9 gene targeting technology;
s20, after gene targeting is finished, recovering for 3 days according to 80% cell density, then inoculating the cells into a 96-well plate according to the density of 0.8 cell per well, and culturing and maintaining by using MSC conditioned medium;
s30, 7 days later, dividing the cells of each well into two parts, one part is continuously cultured, and the other part is rapidly identified based on PCR;
s40, screening positive clones according to the result of rapid identification, and carrying out the next analysis after amplification.
According to the method for rapidly identifying MSC gene modification based on PCR provided by the embodiment of the invention, after gene targeting is finished, cells can be taken for identification only in 10 days, so that the time is greatly saved, and the progress of subsequent work is greatly promoted; moreover, the identification can be completed only by a few cells, which greatly reduces the time of in vitro expansion of the MSC and avoids the function reduction caused by overlong time of in vitro expansion of the MSC. Moreover, compared with the traditional technology, the method screens out positive clones for subsequent work, has short amplification time and less passage times, and also has certain advantages in the aspects of dryness maintenance, differentiation potential and the like.
In some embodiments, the modification of a specific gene of MSC comprises, but is not limited to, integration of the inflammation suppressor IL-10 into the AASV1 genomic site. CRISPR/Cas9 gene editing technology allows for modification of multiple MSC genes, such as inflammatory suppressor IL family members, certain cancer suppressor genes, and the like.
In some embodiments, the specific steps of PCR-based rapid identification include:
s100, rapidly centrifuging the cells at the rotating speed of 5000rpm for 1min, and resuspending the cells with 15 mu l of lysate;
s200, keeping the temperature at 55 ℃ for 60 min;
s300, keeping the temperature at 95 ℃ for 15 min;
s400, quickly centrifuging at the rotating speed of 12000rpm for 1min, and using the obtained supernatant for subsequent PCR identification;
s500, taking the supernatant, adding a specific primer aiming at the modified MSC specific gene, and carrying out PCR amplification;
s600, identifying the size of the PCR amplification product and comparing the size with the size of the expected fragment.
In some embodiments, the lysis solution is 10mM Tris-HCl (pH8.0), 2mM EDTA (pH8.0), 2% Tween 20, and 1mg/ml proteinase K.
The method for rapidly identifying based on PCR provided by the embodiment of the invention can identify the modification of any gene of MSC, and only needs to design a specific primer aiming at the gene and identify the size of a product after PCR amplification. The method is simple and convenient to operate, and has low requirements on operators and instruments; the time consumption is short, and the operation can be completed in a few hours; batch operations are possible, allowing identification of tens or even hundreds of clones at a time.
In some specific embodiments, the specific primer sequence for the AASV1 gene is: a forward primer: 5'-CCCAACCCCATGCCGTCTTCACTCG-3', respectively; reverse primer 5'-CTAGGACGCACCATTCTCACAAAGG-3'.
In some embodiments, positive clones are screened according to the result of the rapid identification, and modified gene expression amount analysis, MSC marker analysis and MSC differentiation ability identification are performed after amplification.
In a second aspect, the embodiments of the present invention further provide an application of a method for rapidly identifying MSC gene modification based on PCR, wherein the method as described in any one of the above is used for identification of MSC gene modification.
The method for rapidly identifying MSC gene modification based on PCR and the application thereof of the present invention are further explained by the following specific examples:
example 1
1. The IL-10 of the inflammation inhibitor is integrated into the AASV1 genome site by using a Cas9 gene targeting technology. FIG. 2 shows a schematic representation of the targeting vector and the targeting site, from which it can be seen that IL-10 is finally inserted in the middle of the two arms of the AASV1 genome.
2. After gene targeting was completed, cells were recovered at a cell density of 80% for 3 days, and then seeded in a 96-well plate at a cell density of 0.8 cells per well, and maintained in culture in MSC conditioned medium.
3. After 7 days, the cells from each well were divided into two, one for continuous culture and the other for rapid PCR-based identification as follows:
a. cells were spun down at 5000rpm for 1min and resuspended in 15. mu.l lysis buffer (10mM Tris-HCl (pH8.0), 2mM EDTA (pH8.0), 2% Tween 20,1mg/ml proteinase K);
b.55 ℃ for 60 min;
c, keeping the temperature at 95 ℃ for 15 min;
d. quickly centrifuging at 12000rpm for 1min to obtain supernatant for subsequent PCR identification;
e. mu.l of the supernatant was taken, 10. mu.M of AASV1 site primer (forward primer: 5'-CCCAACCCCATGCCGTCTTCACTCG-3'; reverse primer: 5'-CTAGGACGCACCATTCTCACAAAGG-3') was added thereto, 25. mu.l of stabilizing 2 XPCR Master Mix (Thermo Scientific Co.) was added thereto, and amplification was carried out for 40 cycles using a PCR instrument;
f. the size of the PCR amplification product was identified by electrophoresis and compared to the expected fragment size, wherein the expected fragment was 1106 bp.
As shown in FIG. 3, the results of the electrophoretic identification revealed that the clones 3, 5, 10, 17, 18, 20, 23, 37, 44 and 54 were positive clones.
4. The positive clones obtained by the above screening were cultured continuously and amplified for further analysis.
Example 2
The positive clones obtained in example 1 were cultured and further analyzed.
1. Identification of positive clones by conventional PCR
The positive clones screened by the rapid identification method are amplified and then identified again by the conventional PCR, and the result is shown in FIG. 4, so that all the screened clones are positive clones, which also proves the reliability of the rapid identification method.
2. Identification of expression quantity of positive clone IL-10 by qRT-PCR
The results of qualitative and quantitative analysis of the expression amounts of IL-10 of the No. 17 positive clone, the No. 16 negative clone and the control obtained by the rapid identification method are shown in FIG. 5, and it can be known that the IL-10 is successfully and highly expressed by the No. 17 positive clone obtained by screening.
3. MSC markers for positive clones by flow analysis
The flow analysis results are shown in fig. 6, and the results show that the MSCs after gene targeting still have the classical molecular markers.
4. Differentiation ability identification of Positive clones
The positive clones obtained by screening were subjected to spontaneous or induced differentiation, and fig. 7 shows that the MSCs after gene targeting still have the ability to differentiate into adipose, osteogenic and chondrocytes, which also confirms that gene targeting did not affect the function of MSCs.
The analysis result shows that the method for rapidly identifying MSC gene modification based on PCR provided by the embodiment of the invention can accurately screen and obtain positive clones and has high reliability. And the function of the cells is not influenced, and the screened MSC still has the stem cell characteristics and the differentiation capability.
In conclusion, the invention provides a method for rapidly identifying MSC gene modification based on PCR and application thereof. The method for rapidly identifying based on PCR provided by the invention can complete identification only by a few cells, and can obtain enough cells for identification only by 10 days at the fastest speed after gene targeting is finished, so that the time is greatly saved, the progress of subsequent work is greatly promoted, and targeted genome modification can be effectively realized in MSC. Moreover, identification only needs few cells, so that the time of in vitro expansion of the MSC is greatly shortened, and the function reduction caused by overlong time of in vitro expansion of the MSC is avoided. Moreover, compared with the traditional technology, the method screens out positive clones for subsequent work, has short amplification time and less passage times, and also has certain advantages in the aspects of dryness maintenance, differentiation potential and the like. In addition, the PCR identification process is simple and convenient to operate, and has low requirements on operators and instruments; the time consumption is short, and the operation can be completed in a few hours; can be operated in batch, allows dozens or even hundreds of clones to be identified at one time, and has extremely high application prospect.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.
Claims (5)
1. A method for rapidly identifying MSC gene modification based on PCR is characterized by comprising the following steps:
A) modifying the specific gene of the MSC by using a Cas9 gene targeting technology;
B) after gene targeting is finished, recovering for 3 days according to 80% cell density, then inoculating the cells into a 96-well plate according to the density of 0.8 cell per well, and culturing and maintaining by using an MSC conditioned medium;
C) after 7 days, the cells from each well were divided into two, one for continuous culture and the other for rapid PCR-based identification;
D) and (4) screening positive clones according to the result of the rapid identification, and carrying out the next analysis after amplification.
2. The method for rapid identification of genetic modifications of MSCs based on PCR as claimed in claim 1, wherein said specific steps of rapid identification based on PCR comprise:
1) cells were rapidly centrifuged at 5000rpm for 1min and resuspended in 15. mu.l of lysate;
2) keeping at 55 deg.C for 60 min;
3) keeping at 95 deg.C for 15 min;
4) quickly centrifuging at 12000rpm for 1min to obtain supernatant for subsequent PCR identification;
5) taking the supernatant, adding a specific primer aiming at the modified MSC specific gene, and carrying out PCR amplification;
6) the size of the PCR amplification product was identified and compared to the expected fragment size.
3. The method for rapid identification of genetic modification of MSC based on PCR of claim 2, wherein the lysis solution comprises 10mM Tris-HCl pH8.0, 2mM EDTA pH8.0, 2% Tween 20 and 1mg/ml proteinase K.
4. The method for rapid identification of MSC gene modification based on PCR as claimed in claim 1, wherein positive clones are selected according to the rapid identification result, and modified gene expression amount analysis, MSC marker analysis and MSC differentiation ability identification are performed after amplification.
5. Use of a method for rapid identification of MSC gene modifications based on PCR, wherein the method according to any of claims 1-4 is used for identification of MSC gene modifications.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111295836.5A CN114134216A (en) | 2021-11-03 | 2021-11-03 | A method and application for rapid identification of MSC gene modification based on PCR |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111295836.5A CN114134216A (en) | 2021-11-03 | 2021-11-03 | A method and application for rapid identification of MSC gene modification based on PCR |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114134216A true CN114134216A (en) | 2022-03-04 |
Family
ID=80392883
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111295836.5A Pending CN114134216A (en) | 2021-11-03 | 2021-11-03 | A method and application for rapid identification of MSC gene modification based on PCR |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114134216A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104471067A (en) * | 2012-05-07 | 2015-03-25 | 桑格摩生物科学股份有限公司 | Methods and compositions for nuclease-mediated targeted integration of transgenes |
| CN108949690A (en) * | 2018-07-17 | 2018-12-07 | 杭州观梓健康科技有限公司 | A method of prepare can real-time detection mescenchymal stem cell bone differentiation cell model |
| CN110747211A (en) * | 2019-10-31 | 2020-02-04 | 北京兴元和济生命医学科技有限公司 | CRISPR-based method for changing expression of gene product and application thereof |
| CN113249456A (en) * | 2021-05-21 | 2021-08-13 | 云南农业大学 | Method for rapidly screening gene editing pig positive cell line |
-
2021
- 2021-11-03 CN CN202111295836.5A patent/CN114134216A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104471067A (en) * | 2012-05-07 | 2015-03-25 | 桑格摩生物科学股份有限公司 | Methods and compositions for nuclease-mediated targeted integration of transgenes |
| CN108949690A (en) * | 2018-07-17 | 2018-12-07 | 杭州观梓健康科技有限公司 | A method of prepare can real-time detection mescenchymal stem cell bone differentiation cell model |
| CN110747211A (en) * | 2019-10-31 | 2020-02-04 | 北京兴元和济生命医学科技有限公司 | CRISPR-based method for changing expression of gene product and application thereof |
| CN113249456A (en) * | 2021-05-21 | 2021-08-13 | 云南农业大学 | Method for rapidly screening gene editing pig positive cell line |
Non-Patent Citations (1)
| Title |
|---|
| WEI JIANG等: "Rapid identification of genome-edited mesenchymal stem cell colonies via Cas9", 《BIOTECHNIQUES》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108359712B (en) | Method for rapidly and efficiently screening SgRNA target DNA sequence | |
| WO2023015759A1 (en) | Adenine base editor fusion protein free of limit by pam, and application | |
| CN107201408B (en) | A method for developing sisal SSR primers based on transcriptome sequencing | |
| Ye et al. | MiniSAGE: Gene expression profiling using serial analysis of gene expression from 1 μg total RNA | |
| CN109593757B (en) | Probe and method for enriching target region by using same and applicable to high-throughput sequencing | |
| CN106868031A (en) | A kind of cloning process of multiple sgRNA series parallels expression based on classification assembling and application | |
| CN105734679B (en) | Nucleic acid target sequence captures the preparation method of sequencing library | |
| CN107893260B (en) | Method and kit for constructing transcriptome sequencing library by efficiently removing ribosomal RNA | |
| CN109055434B (en) | A method for correcting structural mutations in pig KIT gene using CRISPRCas9 technology | |
| CN108424907A (en) | A kind of high throughput DNA multidigit point exact base mutation methods | |
| CN108517567A (en) | Connector, primer sets, kit and the banking process in library are built for cfDNA | |
| WO2023016021A1 (en) | Base editing tool and construction method therefor | |
| Kouprina et al. | Highly selective, CRISPR/Cas9‐mediated isolation of genes and genomic loci from complex genomes by TAR cloning in yeast | |
| WO2023202030A1 (en) | Method for constructing high-throughput sequencing library of small rna | |
| CN113564197A (en) | Construction method and application of CRISPR/Cas 9-mediated plant polygene editing vector | |
| CN114134216A (en) | A method and application for rapid identification of MSC gene modification based on PCR | |
| CN101157934B (en) | Gene clone plasmid based on Phi BT1 integrase and Phi C31 integrase and its uses | |
| CN110241212B (en) | Primer set for sequencing and detecting BRCA1 and BRCA2 gene amplicons and application thereof | |
| CN116042572B (en) | Application of xCas12a protein or related biomaterials | |
| WO2023134719A1 (en) | Construction method for probe hybridization-based high-throughput transcriptional profiling sequencing library | |
| CN114032295A (en) | A kind of human leukocyte antigen ABCDRB1DQB1 genotyping detection method and kit | |
| CN114015724A (en) | Improved CRISPR-Cas9 gene editing method and application | |
| CN112899345A (en) | Construction method of ribosome blot sequencing library | |
| CN113493786A (en) | Method for improving rice grain traits by blocking or weakening expression of OsMIR3979 in rice | |
| CN109943589A (en) | A single base mutation method and system used |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220304 |