Plasmid system for tracing liver precursor cells and application

A technology of liver precursor cells and plasmids, which is applied in the field of medicine and biology, can solve the problems of Epcam+ liver precursor cell research, and achieve the effect of reducing costs

Pending Publication Date: 2022-03-22
中国人民解放军海军军医大学第三附属医院
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the construction of targeted liver precursor cell...

Method used

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  • Plasmid system for tracing liver precursor cells and application
  • Plasmid system for tracing liver precursor cells and application
  • Plasmid system for tracing liver precursor cells and application

Examples

Experimental program
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Embodiment 1

[0035] Embodiment 1 plasmid Epcam-cre-DTR construction and purification

[0036] 1. Construction of expression plasmid pT-Epcam-cre-DTR

[0037] The seamless cloning method is used for construction, and the specific construction steps are as follows:

[0038] After the sequencing is correct, it is digested with Clal and Pmll double enzymes, and connected with the carrier of Clal and Pmll double enzyme digestion. Restriction endonucleases for digestion were purchased from New England Biolabs (NEB), and the reaction was carried out using the system recommended by the enzyme instructions.

[0039] The specific reaction process is as follows: use Clal and Pmll to carry out double enzyme digestion on the above-mentioned PCR product and carrier, and use a high-salt (H) buffer system for the reaction, place it in a 37° C. water bath for 3 hours, and fully digest. After separation by 0.8% agarose electrophoresis, gel recovery was carried out using the gel recovery kit from TIANGEN C...

Embodiment 2

[0056] Example 2 trace verification

[0057] Taking 3,5-diethoxycarbonyl-1,4-dihydrocollidine (3,5-diethoxycarbonyl-1,4-dihydrocollidine, DDC) fed mouse model of liver fibrosis as an example, Epcam- The cre-DTR plasmid system was integrated into the host genome to trace the proliferation of Epcam+ hepatic progenitor cells under liver injury.

[0058] 6 weeks old C57BL / 6J-Rosa26 loxP-stop-loxP–tdTomato Mice were injected with Epcam-cre-DTR targeting plasmid and Sleeping Beauty transposon system ( image 3 A). From week 2 onwards follow the image 3 Feeding DDC in the way shown in B results in bile duct reaction and liver fibrosis in mice and liver damage in mice.

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Abstract

The invention provides a plasmid system Epcam-cre-DTR for tracing liver precursor cells, the base sequence of the plasmid system is as shown in SEQ ID NO.1, and an Epcam promoter sequence, a cre recombinase and a DNA sequence of a diphtheria toxin receptor are inserted into the plasmid system. After an Epcam-cre-DTR plasmid system and a Sleeping Beauty transposon system are subjected to tail intravenous injection, liver precursor cells expressing Epcam are started to specifically generate cre recombinase, the cre recombinase is combined with an loxP site, tdTomato and a diphtheria toxin receptor are expressed, and Epcam + liver precursor cells carry a red fluorescence mark. Through intraperitoneal injection of diphtheria toxin, specific clearance expression of liver precursor cells containing a genome integrated to a host is carried out, and the red fluorescence mark disappears.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, and specifically relates to a plasmid system and a construction method for tracing liver precursor cells and its application in tracing the differentiation and proliferation of liver precursor cells in normal liver homeostasis and damaged liver regeneration. Background technique [0002] The liver is the most regenerative organ in the human body. The homeostasis of normal liver and the source of hepatocytes for regeneration of damaged liver are still controversial issues in the academic circle. Recent studies have shown that self-regeneration of hepatocytes was found in acute and subacute liver injury (Schaub JR, Malato Y, Gormond C, et al. (2014) Evidence against astem cell origin of new hepatocytes in a common mouse model of chronic liverinjury.Cell Rep8(4):933-9.); In chronic liver injury, hepatocytes have been found to be Hnf4α+Sox9+double-positive, showing the ability of self-renewal in...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/62A61K38/16A61K38/17A61K48/00A61K49/00A61P1/16
CPCC12N15/70C07K14/47C12N9/22C07K14/705A61K38/177A61K48/0075A61K48/005A61K38/164A61K49/0045A61K49/0097A61P1/16C07K2319/00Y02A50/30
Inventor 王红阳于乐兴晏梓钧刘傅言
Owner 中国人民解放军海军军医大学第三附属医院
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