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Nanog inhibitory type mutant and uses in Nanog function research

A mutant and functional technology, applied to Nanog inhibitory mutants and its application in Nanog function research, can solve the problem that no one has reported the nuclear entry mechanism.

Inactive Publication Date: 2009-07-29
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As an important transcription factor for maintaining the pluripotency of ES cells, mouse Nanog can only function if it enters the nucleus, but the mechanism of Nanog's nuclear entry has not been reported so far

Method used

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  • Nanog inhibitory type mutant and uses in Nanog function research
  • Nanog inhibitory type mutant and uses in Nanog function research
  • Nanog inhibitory type mutant and uses in Nanog function research

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Culture of pluripotent cell lines

[0036] Mouse pluripotent cell lines (P19 and F9 cells) were cultured on 0.1% gelatin-coated substrates in a medium supplemented with 15% fetal bovine serum FBS (GIBCO) in a 37°C incubator (see Pan, G et al. (2005) J Biol Chem 280(2), 1401-1407).

Embodiment 2

[0037] Embodiment 2: Construction of Nanog vector (SEQ ID NO: 1)

[0038] 2ug of total RNA was reverse transcribed using a reaction system with a final volume of 20ul (see Pan, G et al., (2006) Faseb J 20(10), 1730-1732) to obtain a cDNA library. The mouse Nanog fragment was amplified using the primers in Table 1, and then recovered by agarose gel electrophoresis.

[0039] Table 1: Primers for cloning Nanog fragments:

[0040] SEQ ID NO

[0041] The eukaryotic expression plasmid was constructed by inserting PCR fragments into the multiple cloning site EcoRV of the pCR3.1 vector, that is, the pCR3.1 vector (hereinafter referred to as the vector) was digested by EcoRV at 37 ° C for 4 hours, and then the cDNA from the pluripotent cells The amplified mouse Nanog fragment was ligated in the presence of ligase at 16°C for 4 hours to obtain the eukaryotic expression vector of wild-type mouse Nanog.

Embodiment 3

[0042] Example 3: Construction of mouse Nanog eukaryotic expression vector with nuclear localization signal sequence mutation

[0043] After predicting the amino acid sequence (SEQ ID NO: 5) of the whole mouse Nanog by PSORT II software, we found that there is a nuclear localization signal (Nuclear Localization Signal, NLS) at the amino terminus and carboxyl terminus of its conserved Homeobox domain, namely Nuclear localization signal 1 (NLS1, RKQKMR, SEQ ID NO: 6) and nuclear localization signal 2 (NLS2, RMKCKR, SEQ ID NO: 7). We use molecular biology means (point mutation PCR technology) to perform site-directed mutagenesis using the specific mutation primers in Table 2, and mutate nuclear localization signal 1 and nuclear localization signal 2 into irrelevant sequences-GIQNMQ and GMNVDG, such as figure 1 As shown, the target mutants were obtained, namely 1 mutant mNanog (SEQ ID NO: 8), 2 mutant mNanog (SEQ ID NO: 9) and double mutant mNanog (SEQ ID NO: 10).

[0044] Table ...

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Abstract

The invention relates to inhibitory Nanog mutant which is obtained by changing nuclear localization signals 1 and / or 2 positioned in Nanog conservative structure domain Homeobox (the sequence is seen in SEQ ID NO: 2) to damage the functions, wherein the mutant still has the capability of forming heterodimer with wild type Nanog, and inhibits the function of transcriptional activation of wild type Nanog and the function is brought into play as inhibitory Nanog mutant, thereby being capable of being used as a very useful inhibitory tool to research the function of mouse Nanog.

Description

Technical field: [0001] The present invention relates to the construction and expression of Nanog suppressor mutants and their use in the functional research of Nanog. Said Nanog is a Homeobox family gene, which can maintain the pluripotency of embryonic stem cells independently of LIF. Transcription factor that plays an important role in pluripotency, self-renewal and reprogramming. Background technique: [0002] Embryonic stem cells (ES cells) come from the inner cell mass (ICM) of the mammalian blastocyst (Blastocyst), and are the most primitive stem cells with developmental pluripotency. There are three basic biological characteristics that distinguish ES cells from other cells: First, ES has pluripotency. The so-called pluripotency means that ES cells have multidirectional differentiation potential. First, they can differentiate into internal The cells of the three germ layers of germ layer, mesoderm and ectoderm, and then differentiate into various cell types; second,...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C12N15/79G01N33/68
Inventor 裴端卿张娟张小飞
Owner TSINGHUA UNIV
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