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Method for constructing hESC indicating cell line for specifically tracing endothelial cell differentiation

A technology for indicating cell lines and endothelial cells, which can be used in biochemical equipment and methods, embryonic cells, animal cells, etc., and can solve problems such as lack of regulatory elements, unstable expression, and position effects.

Active Publication Date: 2018-03-16
SHANGHAI PINPOINT MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] ① There are safety issues such as virus integration and immunogenicity in lentivirus mediation;
[0015] ② Lentivirus infection did not obtain site-specific integration clones, and it was a mixed cell, which was not conducive to explaining the problem in subsequent experiments;
[0016] ③Because it is a random integration, there will be problems such as unstable expression or position effect;
[0017] ④ The exogenous CD144 promoter may not contain its adjacent regulatory elements, which will affect gene expression

Method used

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  • Method for constructing hESC indicating cell line for specifically tracing endothelial cell differentiation
  • Method for constructing hESC indicating cell line for specifically tracing endothelial cell differentiation
  • Method for constructing hESC indicating cell line for specifically tracing endothelial cell differentiation

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1 Experimental process

[0045] The primers and probes to be used are listed in Table 1:

[0046] Table 1

[0047]

[0048]

[0049] (1) CD144N-GFP vector construction

[0050] ① Amplify the 5′ and 3′ homology arms, using H9 gDNA as a template:

[0051] The PCR system is as follows:

[0052]

[0053] The thermal cycling conditions are:

[0054]

[0055]

[0056] The primer used for the 5' homology arm is CD144N5F2+CD144N5Rovr, and the product is 831bp.

[0057] The primers used for the 3' homology arm are CD144N3F+CD144N3R1, and the product is 568bp.

[0058] ② Amplify orf+pa of GFP from pEGFP-N1, and add restriction sites of Xba 1 and Nhe 1 in the upstream and downstream respectively. The PCR system is as follows:

[0059]

[0060] The thermal cycling conditions are:

[0061]

[0062] Using CD144N-GFP-ovF+CD144N-GFP-R as primers, the product size is 961bp

[0063] ③ Overlap PCR connects the 5′ homology arm and orf+pa of GFP

[0...

Embodiment 2

[0300] Embodiment 2 experimental result and analysis

[0301] (1) Construction of plasmid CD144N-GFP (see results in figure 1 )

[0302] Using primers CD144N5F2 and CD144N5Rovr to amplify the 5'homologous arm LHA of 831bp; primers CD144N3F and CD144N3R1 amplified the 3'homologous arm SHA of 568bp; amplified GFP by primers CD144N-GFP-ovF and CD144N-GFP-R fragment. LHA and GFP were connected by overlapping primer extension method to form LHA-GFP. SHA and LHA-GFP were cut with PstI and NheI respectively and ligated to form an intermediate vector. The intermediate vector and F8-22-PGK-Neo were respectively digested with NheI enzyme Cut and connect PGK-Neo into the intermediate vector to form the final targeting vector CD144N-GFP.

[0303] (2) Construction of plasmid TALEN (results see figure 2 )

[0304] The TALENs targeting the N-terminus of the CD144 gene, CD144N-L2 and CD144N-R2, were constructed using the "REAL Assembly TALEN Kit" system established by the KeithJoung Lab...

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Abstract

The invention discloses a method for constructing an hESC indicating cell line for specifically tracing endothelial cell differentiation. The method comprises the following steps: (1) constructing a plasmid CD144N-GFP; (2) constructing plasmid TALENs, wherein the plasmid TALENs include a plasmid CD144N-L2 and a plasmid CD144N-R2; and (3) nuclearly transferring the plasmid CD144N-GFP and the plasmid TALENs to ESCs, carrying out G418 screening, picking a monoclone, carrying out PCR preliminary identification by using a cross-5' homologous arm primer F1 / R1 and a cross-3' homologous arm primer F2 / R2, carrying out Southern blot identification on bilaterally positive cells, and carrying out hybridization identification on probes on the long homologous arm by using Kpn I enzyme digestion to obtain positive cells that are the hESC indicating cell line for specifically tracing endothelial cell differentiation.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for constructing an hESC indicator cell line for specifically tracing endothelial cell differentiation. Background technique [0002] Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer unprecedented advantages for disease modeling, drug screening and many other in vitro or in vivo applications. Given their unique therapeutic potential, many studies have developed protocols to efficiently differentiate ESCs into cells of different lineages to avoid teratomas from transplanted undifferentiated ESCs. Therefore, the establishment of lineage-specific reporter ES / iPS cell lines will be essential for a better understanding of the different developmental stages of ES / iPS cells, efficient optimization of differentiation protocols, and in vivo monitoring of transplanted cell survival and cell migration. [0003] Site-specific ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/63
CPCC07K14/43595C07K14/70596C12N5/0606C12N15/63C12N2510/00
Inventor 梁德生胡志青邬玲仟
Owner SHANGHAI PINPOINT MEDICAL TECH CO LTD
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