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CN-121992040-A - Mouse Oct4 conditional gene knockout model construction method based on four-way sgRNA cooperative targeting

CN121992040ACN 121992040 ACN121992040 ACN 121992040ACN-121992040-A

Abstract

The invention relates to the technical field of conditional gene knockout model construction, and discloses a method for constructing a conditional gene knockout model of mouse Oct4 based on four-linkage sgRNA cooperative targeting, which comprises the steps of designing four sgRNAs into sgRNA1, sgRNA2, sgRNA3 and sgRNA4, wherein the sgRNA1 and the sgRNA2 target non-coding regions at the 5 'end and the 3' end of a second exon of an Oct4 gene, the sgRNA3 and the sgRNA4 target structural domain conserved sequences, preparing electrotransfer liquid, comprising Cas9mRNA, the four sgRNAs and electrotransfer buffer liquid, carrying out electrotransfer treatment on fertilized eggs of the mouse, culturing the fertilized eggs subjected to the electrotransfer treatment to a blastula stage, and carrying out genotype identification on the cultured blastula. According to the scheme, the sgRNA combination and embryo electric rotating system are optimized, a mouse Oct4 conditional gene knockout model is efficiently and accurately constructed, a reliable tool is provided for research on the regulation and control mechanism of Oct4 in early embryo three-germ layer differentiation of a mouse, and the medical transformation value of the mouse is mined.

Inventors

  • TANG XIAOHAN

Assignees

  • 重庆医科大学

Dates

Publication Date
20260508
Application Date
20260228

Claims (10)

  1. 1. A method for constructing a mouse Oct4 conditional gene knockout model based on four-way sgRNA cooperative targeting is characterized by comprising the following steps: Step one, designing four sgrnas, namely, sgRNA1, sgRNA2, sgRNA3 and sgRNA4, wherein the sgrnas 1 and 2 target the 5 'end and 3' end non-coding regions of a second exon of an Oct4 gene, and the conserved sequences of the targeting domains of the sgrnas 3 and 4; Step two, preparing electrotransfer liquid which consists of Cas9mRNA, the four sgRNAs and electrotransfer buffer; step three, performing electrotransformation treatment on the fertilized eggs of the mice; Culturing the fertilized eggs subjected to electrotransformation to a blastula stage; and fifthly, carrying out genotype identification on the blastocysts obtained by culture.
  2. 2. The method for constructing the mouse Oct4 conditional gene knockout model based on the four-linked sgRNAs co-targeting of the invention is characterized in that in the first step, the GC content of the 4 sgRNAs is 40-60%, the occurrence of a continuous 4T sequence structure is avoided, and the detection by an MIT_CRISPR design tool ensures that the off-target scores of the 4 sgRNAs are all >90.
  3. 3. The method for constructing the mouse Oct4 conditional gene knockout model based on the four-linked sgRNA cooperative targeting of claim 2 is characterized in that in the first step, the nucleotide sequence of the Oct4 gene is shown as SEQ_ID_NO.1, the nucleotide sequences of the 4 sgRNAs are shown as SEQ_ID_NO.2, the sgRNA2 is shown as SEQ_ID_NO.3, the sgRNA3 is shown as SEQ_ID_NO.4, and the sgRNA4 is shown as SEQ_ID_NO. 5.
  4. 4. The method for constructing the mouse Oct4 conditional gene knockout model based on the four-linked sgRNA cooperative targeting of claim 1, wherein in the second step, the electrotransfer solution comprises 50 ng/μl of Cas9_mRNA, 25 nM of 4 sgRNAs and 20% of electrotransfer buffer solution with final concentration of 100 mM of HEPES, 750 mM of KCL, 5mM of MgCl 2 ·6H 2 O, 5mM of TCEP, 50% of glycol, pH of 7.5 adjusted by KOH and the balance of ddH 2 O.
  5. 5. The method for constructing the mouse Oct4 conditional gene knockout model based on the four-linked sgRNA cooperative targeting of claim 1 is characterized in that in the third step, the condition of the electrotransformation treatment is that the voltage is 30V, the pulse is 4-6 times, the pulse length is 3ms, and the pulse interval is 100ms.
  6. 6. The method for constructing the mouse Oct4 conditional gene knockout model based on the four-tandem sgRNA cooperative targeting according to claim 1, wherein in the fourth step, the culture is performed in a constant temperature incubator with 37 ℃ and 5% CO 2 until the development of the mouse Oct4 conditional gene knockout model reaches a blastula stage.
  7. 7. The method for constructing the mouse Oct4 conditional gene knockout model based on the four-linked sgRNA cooperative targeting of claim 1 is characterized in that in the fifth step, the genotyping comprises (1) primary screening by amplifying a second exon editing region of an Oct4 gene through nested PCR, (2) deep verification by ChIP-Seq sequencing combined with 0.025 restriction enzyme detection, and (3) off-target analysis by predicting off-target sites through whole genome sequencing detection.
  8. 8. The method for constructing the mouse Oct4 conditional gene knockout model based on the four-tandem sgRNA cooperative targeting of claim 7, wherein the primers used in the primary screening comprise an outer primer F1/R1 and an inner primer F2/R2, the nucleotide sequence of the outer primer F1 is shown as SEQ ID NO.6, the nucleotide sequence of the outer primer R1 is shown as SEQ ID NO.7, the nucleotide sequence of the inner primer F2 is shown as SEQ ID NO.8, and the nucleotide sequence of the inner primer R2 is shown as SEQ ID NO. 9.
  9. 9. The mouse Oct4 conditional gene knockout model is characterized in that the oosperm of the mouse Oct4 conditional gene knockout model is constructed by the method of any one of claims 1-8 until the oosperm rate is more than or equal to 85%, the gene editing efficiency is more than or equal to 85%, and the off-target rate is less than or equal to 1.5%.
  10. 10. The use of a conditional gene knockout model of mouse Oct4 according to claim 9 for studying the dynamic regulatory mechanism of early embryonic trigermal differentiation in mice to assess the risk of embryonic lethality associated with mutation of human homologous gene Oct 4L.

Description

Mouse Oct4 conditional gene knockout model construction method based on four-way sgRNA cooperative targeting Technical Field The invention relates to the technical field of conditional gene knockout model construction, in particular to a method for constructing a mouse Oct4 conditional gene knockout model based on four-way sgRNA cooperative targeting. Background The construction of the mouse Oct4 conditional gene knockout model is a core tool for analyzing the Oct4 gene function, exploring embryo development mechanism and promoting regeneration medical research. Oct4 is used as a key transcription factor of POU family, is a core molecule for maintaining embryo stem cell multipotency and regulating early embryo development, and is widely involved in the characteristics regulation and somatic reprogramming process of cell clusters in blastula, and the dysfunction is closely related to embryo dysplasia and stem cell differentiation disorder. The embryo mortality of the traditional Oct4 full-knockout model is extremely high, a living individual is difficult to obtain, the conditional gene knockout model can realize space-time specific knockout of the Oct4 gene in a specific germ layer and a specific development stage, effectively avoids the problem of embryo death, accurately analyzes the dynamic regulation and control function of the model, plays an irreplaceable important role in the research of the molecular mechanism related to Oct4 and the application of regenerative medical transformation, and provides reliable animal model support for the research of pathogenesis and targeted treatment of related diseases. In the prior art, in order to improve the construction efficiency and precision of a mouse Oct4 conditional gene knockout model, a plurality of technical paths are formed around gene editing technology optimization, flow improvement and risk control, namely, firstly, the traditional homologous recombination technology is optimized, the model construction precision is improved by improving a vector construction strategy and optimizing a screening method, the model construction period is shortened, secondly, CRISPR/Cas9 gene editing technology is used for replacing the traditional homologous recombination technology, the operation flow is simplified, the targeting editing efficiency is improved by optimizing sgRNA design and adjusting a Cas9 protein delivery mode, thirdly, the targeting specificity is improved by adopting a single sgRNA or double sgRNA mediated editing strategy, the off-target risk is reduced, fourthly, a model screening and verification system is perfected, the model construction precision is improved, the influence of nonspecific variation on the model accuracy is reduced by the steps of genotype identification, off-target detection and the like, and fifthly, embryo injection and implantation technology is optimized, the embryo survival environment is improved, the embryo mortality is reduced, and the individual survival model acquisition rate is improved. However, the existing technology for improving the construction efficiency and precision of the mouse Oct4 conditional gene knockout model still has the technical problems that firstly, the construction efficiency is low, the period is long, the construction period of the traditional homologous recombination technology is as long as 12-18 months, the positive cloning rate is less than 10%, even though the traditional CRISPR/Cas9 technology is limited by single sgRNA design, the editing efficiency fluctuation is larger (25-75%), the stable and efficient large-scale model construction cannot be realized, secondly, the editing precision is insufficient, the off-target risk is high, the average off-target rate reaches 5-20% when the traditional CRISPR/Cas9 system is applied to Oct4 gene editing, the off-target induced nonspecific gene variation can destroy the model accuracy, the accurate analysis of the Oct4 gene function is interfered, thirdly, the embryo survival capability is weak, the specific knockout is difficult to realize, the conditional knockout model is not effective in time-space specific, the specific gene can not be realized, the specific phase is difficult to realize, the specific gene can not realize the accurate regulation and control method is difficult to realize, the dynamic gene knockout model is difficult to realize the accurate analysis by the accurate and the dynamic gene knockout model is difficult to realize, the accurate and the dynamic gene knockout model is difficult to realize by verifying the dynamic gene knockout model has the accurate analysis, the mechanism is difficult to realize when the dynamic gene knockout model is applied to the dynamic gene has the dynamic gene knockout mechanism is difficult to be completely and has the dynamic gene has the detection and has the effect of detecting and has the influence on the gene analysis on the accuracy and has the accuracy of the gene analysis. In summary, t