CN114958907A - Method for constructing Bcl6 gene super enhancer knockout animal model based on CRISPR/Cas9 technology and application - Google Patents
Method for constructing Bcl6 gene super enhancer knockout animal model based on CRISPR/Cas9 technology and application Download PDFInfo
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Abstract
The invention discloses a method for constructing a Bcl6 gene super enhancer knockout animal model based on a CRISPR/Cas9 technology and application thereof. The construction method of the Bcl6-SE knockout animal model comprises the following steps: 1) gRNA design targeting mouse Bcl6-SE sequence: 2) purifying the pair of gRNAs in the step 1) and injecting the gRNAs and cas9 plasmid into a mouse embryo to obtain an F0 generation mouse; 3) identifying Bcl6-SE genotype, and screening out positive F0 generation mice; 4) and hybridizing the positive F0-generation mouse with a wild mouse to obtain an F1-generation mouse, and selfing the F1-generation heterozygous mouse to obtain a homozygous progeny so as to construct an animal model with the Bcl6-SE gene knocked out. The method realizes the site-directed knockout of the Bcl6-SE regulatory sequence based on the CRISPR/Cas9 technology, and has the advantages of simple operation and high knockout efficiency.
Description
Technical Field
The invention belongs to the technical field of experimental animal models of bioengineering, and particularly relates to a method for constructing a Bcl6 gene super enhancer knockout animal model based on a CRISPR/Cas9 technology and application thereof.
Background
The Bcl6(B cell lymphoma 6) gene encodes a transcription repressing factor containing a conservative zinc finger structure, and belongs to the BTB/POZ transcription factor family. Bcl6 inhibits the expression of many target genes by recruiting specific chromatin modification co-suppression complexes. Bcl6 was originally identified as a proto-oncogene, playing a key role in humoral immunity and lymphomatosis. Bcl6 can mediate abnormal proliferation, genomic instability, and differentiation arrest of diffuse large B-cell lymphoma (DLBCL) cells. Germinal Center (Germinal Center) is where B cells produce high affinity antibodies, while Bcl6 is a key regulator of Germinal Center. Bcl6 also plays a role in early B cell development and may counteract apoptosis induced by immunoglobulin light chain V (D) J recombination.
Super Enhancers (SEs) are super enhancers formed by serially connected regulatory elements, which can strongly drive the expression of cell-associated genes, and hundreds of SEs in cells control key genes and determine the fate trend of cells. In tumorigenesis, senile dementia, diabetes and many autoimmune diseases, it is found that the expression of the pathogenic gene is highly correlated with the abnormal activation of part of the super enhancer. Research shows that a super enhancer (Bcl6-super enhancer, Bcl6-SE) exists near Bcl6, and the enhancer has a certain regulation effect on Bcl6, so that the enhancer is involved in the development regulation of T cells. However, how Bcl6-SE regulates the expression of Bcl6 gene is still unclear, so that the process of influencing T cell development is still unclear, and the construction of a super enhancer Bcl6-SE knock-out mouse is particularly important.
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the related new generation gene editing technology mediated by the Cas9 protein have the advantages of easy operation and low cost, can realize accurate and efficient gene editing functions, including site-specific insertion, site-specific knockout, site-specific mutation and the like, and are widely used for gene editing of various animals and plants. Between the advantages of gene editing of CRISPR/Cas9, the technology is adopted to carry out gene editing on Bcl6-SE, and a super enhancer knock-out mouse model is constructed.
Disclosure of Invention
The invention aims to provide a method for constructing a Bcl6 gene super enhancer knockout animal model based on a CRISPR/Cas9 technology and application thereof.
Aiming at the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, a method for constructing a Bcl6 gene super enhancer knockout animal model based on a CRISPR/Cas9 technology comprises the following steps:
1) gRNA design targeting C57 mouse Bcl6-SE sequence:
designing a pair of corresponding gRNAs according to a Bcl6-SE sequence, wherein the sequences of the gRNAs are shown as SEQ ID NO.1 and SEQ ID NO. 2;
2) mRNA preparation and microinjection F0 mice were obtained:
purifying the pair of gRNAs in the step 1) and injecting the gRNAs and cas9 plasmid into a mouse embryo to obtain an F0 generation mouse;
3) identifying Bcl6-SE genotype, and screening out positive F0 generation mice;
4) and hybridizing the positive F0-generation mouse with a wild mouse to obtain an F1-generation mouse, and selfing the F1-generation heterozygous mouse to obtain a homozygous progeny so as to construct the Bcl6-SE knockout animal model.
Further, the primers used for Bcl6-SE gene identification in the step 3) comprise a common upstream primer and two downstream primers, wherein the sequence of the upstream primer is shown as SEQ ID NO.3, and the sequence of the downstream primer is shown as SEQ ID NO.4 and EQ ID NO. 5.
In a second aspect, the invention provides an animal model of Bcl6-SE knockout constructed by the above method.
In a third aspect, the Bcl6-SE knockout animal model constructed by the invention can be applied to research on the regulation and control of Bcl6 gene expression by a super enhancer in the T cell development process.
Compared with the prior art, the invention has the following beneficial effects:
the site-directed knockout of Bcl6-SE is realized based on the CRISPR/Cas9 technology, the method has the advantage of high knockout efficiency, and the constructed Bcl6-SE knockout animal model can have the function research of a super enhancer in the T cell development process, thereby being beneficial to explaining the mechanism of T cell development and providing a foundation for the treatment of immune system diseases.
Drawings
FIG. 1 is a structural diagram of mouse Bcl 6-SE;
FIG. 2 is a diagram of the design scheme of primers for Bcl6-SE genotyping;
FIG. 3 is an agarose electrophoresis picture of genotype determination of F0 mouse;
FIG. 4 is a graph showing the result of sequencing and identification of F0 mouse.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
Example 1
A method for constructing a Bcl6-SE knockout animal model based on a CRISPR/Cas9 technology comprises the following steps:
1. gRNA design targeting mouse Bcl6-SE regulatory sequences
A pair of gRNAs (gRNA1 and gRNA2) for Bcl6-SE gene knockout are designed at corresponding positions on both sides of a Bcl6-SE region, wherein the positions of the gRNA1 and the gRNA2 are shown in FIG. 1, and the sequences are shown in Table 1. Wherein, the nucleotide sequence of the targeting Bcl6-SE is positioned between a Bcl6 gene and an Lpp gene, and the gRNA1 and the gRNA2 are respectively positioned at the upstream and the downstream of the interval.
TABLE 1 gRNA sequences for mouse Bcl6-SE sequence knockdown
| gRNA name | SEQ ID NO | gRNASequence(5'to3') |
| gRNA1 | SEQ ID NO.1 | TTAAAGTAGCTTCGCTCATC |
| gRNA2 | SEQ ID NO.2 | CTAAGGCTGGGCATTCGGAT |
mRNA preparation and microinjection to obtain F0 mice
Purifying the gRNA1 and the gRNA2 in the step 1), injecting a C57 mouse embryo together with a Cas9 plasmid (the Cas9 plasmid is obtained by transformation in a laboratory of an inventor), transplanting the embryo into an oviduct of a surrogate recipient mouse after injection, and obtaining 284 embryos by prokaryotic injection.
Birth and identification of F0 mouse
The number of piglets was 38, and the F0 mice were subjected to tail-clipping identification 1 week after birth to give 6 positive (2 females, 4 males) F0 mice with black hair color as shown in table 2.
TABLE 2 information List of positive F0 mouse generations
| Date of birth | Number of | Number and sex |
| 2018/6/4 | 2 | ♂1257、1260 |
| 2018/6/25 | 4 | ♀1482、1499♂1489、1491 |
Specific primer design scheme for Bcl6-SE genotype identification is shown in FIG. 2, wherein a common upstream primer is adopted by a Wild Type (WT) and a Bcl6-SE gene knock-out type (KO), the upstream primer is adopted by the wild type by Bcl6-SE-seqF, and the downstream primer is adopted by the Bcl6-SE-seqR 1; the Bcl6-SE gene knockout type adopts Bcl6-SE-seqF as an upstream primer and Bcl6-SE-seqR2 as a downstream primer. The sequences of Bcl6-SE-seqF, Bcl6-SE-seqR1 and Bcl6-SE-seqR2 are shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 in Table 3, respectively.
TABLE 3 specific primers for SEts2 genotype identification
| Primer | SEQ ID NO | Sequence(5'to3') | Description of the invention |
| Bcl6-SE-seqF | SEQ ID NO.3 | TGGATTTCCAGGCGTACTTC | Common upstream primer |
| Bcl6-SE-seqR1 | SEQ ID NO.4 | GAGAGGCCCTTGTTCATGTG | WT special downstream primer |
| Bcl6-SE-seqR2 | SEQ ID NO.5 | TGGGCTAGAGATGTTTAGGG | Downstream primer special for KO |
The PCR reaction system and the reaction program for carrying out tail-cutting identification on the F0 mouse after 1 week of birth are shown in tables 4 and 5, the sizes of PCR products aiming at the Bcl6-SE gene in a wild type and a Bcl6-SE gene knockout type after PCR reaction are 446bp and 503bp respectively, wherein the standard for carrying out genotype judgment on the F0 mouse is shown in table 6, 446bp products are obtained after PCR amplification is carried out on the Bcl6-SE-seqF and the Bcl6-SE-seqR1, and no product is obtained after PCR amplification is carried out on the Bcl6-SE-seqF and the Bcl6-SE-seqR2, and the mouse is determined to be the wild type mouse; when no product is obtained after PCR amplification is carried out on Bcl6-SE-seqF and Bcl6-SE-seqR1, and a 503bp product is obtained by PCR amplification on Bcl6-SE-seqF and Bcl6-SE-seqR2, the mouse is determined to be a SEts2 gene knockout type; when 446bp products are obtained by PCR amplification of Bcl6-SE-seqF and Bcl6-SE-seqR1, and 503bp products are obtained by PCR amplification of Bcl6-SE-seqF and Bcl6-SE-seqR2, the mice are heterozygous, namely positive F0 generation mice.
TABLE 4 PCR reaction System
| Reagent | Volume of |
| 2xPCR mix | 10μL |
| Bcl6-SE-seqF | 0.5μL |
| Bcl6-SE-seqR1 or Bcl6-SE-seqR2 | 0.5μL |
| Template gDNA | 1μL |
| Ultrapure water | 8μL |
TABLE 5 PCR reaction procedure
TABLE 6 mouse genotype criteria
| Genotype(s) | Bcl6-seqF+Bcl6-seqR1 | Bcl6-seqF+Bcl6-seqR2 |
| Wild type WT (+/+) | 446bp | no product |
| Other Heter (+/-) | 446bp | 503bp |
| SEts2 knock-out type KO (-/-) | no product | 503bp |
FIG. 3 shows the agarose electrophoresis pattern for genotyping of F0 mouse Bcl6-SE, wherein 38 born mice were subjected to PCR and agarose electrophoresis detection, and six mice, numbered 1257, 1260, 1482, 1499, 1489 and 1491, were used as gene-editing mice. Direct sequencing is carried out on the obtained six gene editing mice, Bcl6-SE knockout identification is carried out on F0 generation mice, the sequencing result is shown in figure 4, the genotypes of 1257, 1260, 1482 and 1489 four mice are the same, and the knockout length is 119.2 kb; the 1491 mouse knockout length is 119.2 kb; the 1499 mouse knockout length is 119.2kb, which indicates that the Cas9 gene knockout mediated by gRNA1 and gRNA2 constructed by the invention can effectively knock out Bcl6-SE fragments.
And 4, breeding the F0 generation positive mice by using No. 1257, breeding the F0 generation positive mice with wild C57 mice after sexual maturity of 8 weeks, and carrying out tail-cutting identification on the F1 generation mice at 1 week of age. 5F 1 mice were born, and 4 positive F1 mice were genotyped. Information from the F1 mouse is shown in Table 7.
TABLE 7 reproductive information of F0 and C57 mice
| Parent strain | F1 date of birth | Number of | Number and sex of F1 positive mice |
| ♀C57 X♂1257 | 2018/10/8 | 4 | ♀5596、5597、5598♂5599 |
TABLE 8F 1 passage positive mouse information
The method for constructing the Bcl6-SE knockout animal model has the advantages of simple operation and high knockout efficiency; the Bcl6-SE knockout animal model constructed by the method is helpful for researching a Bcl6-SE gene regulation mechanism in which a super enhancer participates.
Claims (4)
1. A method for constructing a Bcl6 gene super enhancer (Bcl6-SE) knockout animal model based on a CRISPR/Cas9 technology is characterized by comprising the following steps of:
1) gRNA design targeting mouse Bcl6-SE sequence:
designing a pair of corresponding gRNAs according to a Bcl6-SE sequence, wherein the sequences of the gRNAs are shown as SEQ ID NO.1 and SEQ ID NO. 2;
2) gRNA preparation and microinjection yielded F0 mice:
purifying the pair of gRNAs in the step 1), and injecting a mouse embryo together with cas9 plasmid to obtain an F0 generation mouse;
3) identifying Bcl6-SE genotype, and screening out positive F0 generation mice;
4) and hybridizing the positive F0-generation mouse with a wild mouse to obtain an F1-generation mouse, and selfing the F1-generation heterozygous mouse to obtain a homozygous progeny so as to construct the Bcl6-SE knockout animal model.
2. The method for constructing the Bcl6-SE knockout animal model according to claim 1, wherein the primers used in the Bcl6-SE genotyping identification in the step 3) comprise a common upstream primer and two downstream primers, wherein the sequence of the upstream primer is shown as SEQ ID No.3, and the sequence of the downstream primer is shown as SEQ ID No.4 and EQ ID No. 5.
3. An animal model of Bcl6-SE knock-out constructed by the method of claim 1 or 2.
4. The animal model of claim 3 wherein the mechanism of super enhancer regulation of the Bcl6Bcl6 gene can be studied during T cell development.
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