Dual-homologous recombination pedigree tracing technique

A recombinase and genetic recombination technology, applied in the field of double homologous recombination lineage tracing, can solve the problem that the tracing results are not necessarily accurate

Active Publication Date: 2018-03-09
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Cre may not be absolutely specifically expressed in the targeted tracking cells, but the expression of Cre in the non-targeted tracking cells causes the so-called ectopic expression, and the tracking results of this ectopic expression of Cre may not be accurate ,as the picture shows( figure 1 b) If the inventor believes that Cre is mainly expressed specifically in A cells, then the expression of Cre in...

Method used

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  • Dual-homologous recombination pedigree tracing technique
  • Dual-homologous recombination pedigree tracing technique
  • Dual-homologous recombination pedigree tracing technique

Examples

Experimental program
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Effect test

Embodiment 1

[0156] Example 1. Construction and verification of mouse DR1, a double homologous recombination lineage tracing system tool based on Cre-loxP and Dre-rox

[0157] There are two key factors in the Cre-loxP-based lineage tracing system, which are the expression profile of Cre and the reporter gene system containing loxP sites. Therefore, improving the existing lineage tracing system can start from these two aspects. Since it is relatively difficult to obtain an accurate expression profile of Cre, the research focus of the present invention is mainly on improving the existing reporter gene system containing loxP sites. Since there is a relatively difficult problem in the existing lineage tracing system, that is, when Cre is ectopically expressed (that is, Cre is considered to be expressed in type A cells in theory, but in fact Cre is also expressed in other types of cells) Medium and difficult to be detected), the reliability of the experimental results is greatly reduced, which...

Embodiment 2

[0160] Example 2. The double homologous recombination system can realize the selective and preferential occurrence of Cre-loxP or Dre-rox to block the occurrence of another homologous recombination reaction

[0161] In order to be able to select Cre-loxP or Dre-rox to occur preferentially in the DR1 double homologous recombination system of the present invention, the inventors simultaneously combined the continuous and inducible homologous recombination system with DR1 (such as x-Dre; y-CreER; DR1 or x-Cre; y-DreER; DR1), in theory, in this system, when a continuous homologous recombination reaction (such as Cre-loxP) occurs, then the inducible homologous The recombination reaction (DreER-rox) will no longer be able to take place in the same cell. In order to verify this theory, the inventors first used the existing tool mice of coronary vascular endothelial cells (Tie2-Cre and Apln-DreER) as research objects, and Tie2-Cre mainly marked endocardial cells and coronary vascular ...

Embodiment 3

[0162] Example 3. Using double homologous recombination system to solve the problem of heterotopic homologous recombination

[0163] Next, the inventors applied the above-mentioned double homologous recombination system strategy to the ectopic homologous recombination reaction of blocking cells ( figure 1 b). An important scientific issue that is still controversial in the cardiovascular field is whether the so-called Kit exists in the adult heart. + Stem cells differentiate into cardiomyocytes, and the main focus of controversy is the specific expression of Cre driven by Kit. Currently About Kit + The conclusion that stem cells can become cardiomyocytes is mainly based on the genetic lineage tracing results of Kit-CreER. Interpretation of tracer results will no longer be reliable. There are two possibilities here, one is that there is indeed a Kit + Stem cells differentiate into cardiomyocytes, and the second is that there is no so-called Kit + Stem cells differentiate ...

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Abstract

The invention provides a dual-homologous recombination pedigree tracing technique, and particularly provides a dual-homologous recombination system. In the system, LoxP site and Rox site exist in an embedding manner. By virtue of the system, the other homologous recombination reaction can be prevented by preliminarily selecting one homologous recombination reaction in the existence of Cre recombinant enzyme or Dre recombinant enzyme. By utilizing the dual-homologous recombination system, the genetic recombination operation can be performed for micro organisms, plants and animals.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a double homologous recombination lineage tracing technique and its application. Background technique [0002] Genetic lineage tracing is an effective means to study development, disease and regeneration. This technology mainly utilizes the Cre-LoxP site-specific recombination system of P1 phage, which consists of Cre homologous recombinase and LoxP site, and Cre can specifically recognize LoxP site, but not other sites. Cre and LoxP do not exist in mammalian cells, so the Cre-LoxP system can be introduced into mammals. When two LoxP sites in the same direction are located on both sides of the transcription termination sequence, the termination sequence will be removed in cells expressing Cre, and the reporter gene located behind the termination sequence can be continuously expressed (if the transcription of this site is continuously open, such as Rosa26 site...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/85A01K67/027
CPCA01K67/0275A01K2227/105A01K2267/03C12N15/63C12N15/85C12N2800/30
Inventor 周斌何灵娟李燕蒲文娟
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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