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Construction and application of KRT10 site-directed gene knock-in P2A-CrePR1-T2A-tdTomato mouse model

A KRT10, gene knock-in technology, applied in the field of bioengineering

Pending Publication Date: 2022-01-07
THE FIRST PEOPLES HOSPITAL OF FOSHAN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows researchers to create specific types of animals that have certain functions or characteristics during healing processes such as tissue regeneration after injury. These experiments involve injecting small molecules into animal models followed by monitoring their behavior over time under different conditions like temperature changes or stress stimuli. By analyzing these data from this experiment, scientists are able to better understanding how they work towards developing new treatments for skin disorders caused by trauma or disease.

Problems solved by technology

This patented describes how certain types of cells called Keratins can help keep your body healthy by providing supportive tissue layers that make it harder or easier to break down when needed again later during recovery after injury.

Method used

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  • Construction and application of KRT10 site-directed gene knock-in P2A-CrePR1-T2A-tdTomato mouse model
  • Construction and application of KRT10 site-directed gene knock-in P2A-CrePR1-T2A-tdTomato mouse model
  • Construction and application of KRT10 site-directed gene knock-in P2A-CrePR1-T2A-tdTomato mouse model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1, verifying the DNA sequence of the constructed strain mouse.

[0051] Select C57BL / 6J mice as donor mice, using the primer pair: EGE-STY-061-MSD-F:5'-GCATCAAGCTTGGTACCGATGCTGAAACTA-3'(SEQ ID NO.11) and EGE-STY-061-MSD-R : 5'-ACTTAATCGTGGAGGATGATCTCATACTGGT-3' (SEQ ID NO.12), carry out PCR amplification and perform PCR amplification and sequencing verification on the mouse tail target site sequence of C57BL / 6J, to ensure that the DNA sequence of the constructed strain mouse can be compared with The subsequent design of the sgRNA recognition sequence is compatible.

[0052] After sequencing, it was confirmed that the length of the amplified product of this example was 763bp, which was completely consistent with the DNA sequence of C57BL / 6J mice.

Embodiment 2

[0053] Embodiment 2, design and acquisition of sgRNA.

[0054] According to the target site sequence known in Example 1, 7 potential sgRNAs were designed, which are:

[0055] sgRNA1: 5'-AGTGATCAGGACGATTATTGAGG-3' (SEQ ID NO.11);

[0056] sgRNA2: 5'-AGTGATCAGGACGATTATTGAGG-3' (SEQ ID NO.12);

[0057] sgRNA3: 5'-AGTCTCTTCATACGGGCCACTGG-3' (SEQ ID NO.1);

[0058] sgRNA4: 5'-GACGAAAGGACTCTACCCTCAGG-3' (SEQ ID NO.13);

[0059] sgRNA5: 5'-AATAATCGTCCTGATCACTTTGG-3' (SEQ ID NO.14);

[0060] sgRNA6: 5'-CATAGAAGAGTCTCTTCATACGG-3' (SEQ ID NO.15);

[0061] sgRNA7: 5'-TACTAACAAGCTATTACAAA AGG-3' (SEQ ID NO. 16).

[0062] Biocytogen’s self-developed CRISPR / Cas9 activity detection method-UCATM method was used to detect the activity of sgRNA, and the detection results were as follows: figure 2 As shown, the activity of sgRNA3 is the highest, and the sequence of sgRNA3 is used as sgRNA for subsequent construction.

[0063] The sgRNA obtained by screening was connected to a plasmid vec...

Embodiment 3

[0064] Embodiment 3, construction Cas9 / sgRNA plasmid.

[0065] Synthesize oligos (oligonucleotides) through the sgRNA sequence designed in Example 2, and then connect the sgRNA into the pCS vector by annealing and polymerization, and the ligation product will be sent for correct sequencing after conversion; wherein the size of the Cas9 / sgRNA plasmid vector is 9918bp, the map is as follows image 3 shown.

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Abstract

The invention discloses construction and application of a KRT10 site-directed gene knock-in P2A-CrePR1-T2A-tdTomato mouse model. A construction method comprises the following steps: preparing sgRNA, and constructing a Cas9/sgRNA plasmid; constructing a recombinant targeting vector in which a KRT10 fixed-site gene is knocked into P2A-CrePR1-T2A-tdTomato; injecting the Cas9/sgRNA plasmid and the recombinant targeting vector into a fertilized egg of a donor mouse to obtain a transfected fertilized egg; transplanting the transfected fertilized egg into a uterus of a pseudopregnant female mouse so as to birth F0-generation mice, carrying out genotype identification on the F0-generation mice, and taking mice with positive homologous recombination as chimeric mice; hybridizing the chimeric mice with wild type mice, carrying out genotype identification on the offspring, and taking mice with positive homologous recombination as F1-generation mice; and hybridizing the F1-generation mice, and identifying that descendant mice which are correctly recombined and are not randomly inserted are mouse models.

Description

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Claims

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Application Information

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Owner THE FIRST PEOPLES HOSPITAL OF FOSHAN
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