Application of transcription factor sp1 in regulating the expression of porcine rtl1 gene

A transcription factor and gene expression technology, applied in the field of genetic engineering, achieves the effect of careful design, reliable results and good application prospects

Active Publication Date: 2021-11-09
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the regulation of RTL1 gene

Method used

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  • Application of transcription factor sp1 in regulating the expression of porcine rtl1 gene
  • Application of transcription factor sp1 in regulating the expression of porcine rtl1 gene
  • Application of transcription factor sp1 in regulating the expression of porcine rtl1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Determination of the core promoter region of the pig RTL1 gene of embodiment 1

[0030] 1. Biological information analysis of the promoter region of porcine RTL1 gene

[0031] According to the sequence of the porcine RTL1 gene in NCBI, primers RPF1 (as shown in SEQ ID NO: 9) and RPR1 (as shown in SEQ ID NO: 10) were designed, and the 5' upstream sequence of the RTL1 gene including its first exon was obtained by cloning. A part of the nucleotide sequence with a total of 1475bp (-1394 / +81) (as shown in SEQ ID NO: 8).

[0032] Using the online website (http: / / www.fruitfly.org / seq_tools / promoter.html) to predict and find that the segment from -1027bp to -978bp may be the core promoter region of the gene.

[0033] 2. Primer design

[0034] For further verification, using the 5' upstream sequence of the RTL1 gene obtained above as a template, Primer5 software was used to design 5 upstream deletion primers (P1F-P5F) for amplifying the 5 deletion fragments (P1-P5) in the prom...

Embodiment 2

[0052] Example 2 Determination of the combination of transcription factor SP1 and RTL1 gene core promoter

[0053] 1. Biological information analysis

[0054] Using the TESS website (http: / / www.cbil.upenn.edu / cgi-bin / tess / tess) and

[0055] The TFSEARCH (http: / / www.cbrc.jp / research / db / TFSEARCH.html) website predicts potential transcription factor binding sites in the promoter region, and found that the transcription factor SP1 binding site with a higher score is located at (-247bp / -239bp).

[0056] 2. Co-immunoprecipitation

[0057] The combination of transcription factor SP1 and RTL1 promoter in vivo was detected by chromatin immunoprecipitation (ChIP) assay of PK15 cells. The EZ-ChIPTM Chromatin Immunoprecipitation Kit kit from Millipore, USA was used, and the specific operation process was referred to the instructions of the kit. The main steps are:

[0058] (1) Cultivate and collect PK15 cells for later use, process the genome of PK15 cells by ultrasonic disruption, ...

Embodiment 3

[0070] Example 3 Activity Detection of Transcription Factor SP1 Binding Site Mutant Luciferase Reporter Carrier

[0071] 1. Construction of mutant vector SP1-mut

[0072] Using the wild-type P2 vector as a template, the transcription factor SP1 binding site mutation vector SP1-mut was constructed by using the recombinant PCR rapid gene site-directed mutagenesis method.

[0073] 1. Design of binding site mutation primers

[0074] Using the wild-type P2 vector as a template, design mutation primers for the SP1 binding site GGGGCGGGG as follows, the bases after mutation are underlined, the upstream and downstream primers are reverse complementary, and there is a sequence overlap of 47 bp. The upstream primer (SP1-mut-F) and downstream primer (SP1-mut-R) are as follows:

[0075] table 3

[0076]

[0077] 2. Amplification of overlapping fragments

[0078] Use primer P2F as the upstream primer, SP1-mut-R as the downstream primer to amplify fragment F1, use SP1-mut-F as the up...

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Abstract

The invention provides the application of transcription factor SP1 in regulating the expression of pig RTL1 gene. The present invention takes RTL1 as the research object, by constructing the recombinant plasmid of porcine RTL1 gene promoter dual luciferase reporter gene, and finding the core promoter region of RTL1; and verifying the interaction between the transcription factor SP1 and the RTL1 core promoter region; Then, the SP1 overexpression vector and synthetic small interfering RNA were constructed to detect the effect of SP1 on RTL1; the transcription factor SP1 binding site mutation fluorescent expression vector SP1-mut was also constructed to detect luciferase activity after transfection into PK15 cells and C2C12 cells. The invention confirms the influence of transcription factor SP1 on RTL1 gene transcriptional regulation for the first time, and brings a deeper level of cognition to the expression regulation of RTL1. It is applied to the improvement of livestock meat quality traits and the research on the molecular regulation mechanism of muscle development, and has good application prospect.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the application of transcription factor SP1 in regulating the expression of pig RTL1 gene. Background technique [0002] RTL1 gene (retrotransposon-like-1), also known as paternally expressed gene 11 (Peg11), is an imprinted gene expressed by paternal parents in humans and mice, without Intron sequence. The RTL1 gene is highly expressed in the embryo and placenta, and plays an important role in the nutrient transfer between the mother's placenta and the fetus. Mice knocked out of this gene show increased fetal mortality, neonatal growth arrest, and small placental size ; It is also closely related to human fetal development and neonatal growth. The gene is located in the imprinted region of DLK1-DIO3, and the gene in this region has a certain influence on muscle development. Ectopic expression of RTL1 gene in transgenic mice will lead to muscle hypertrophy; in addit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12N15/113C12N15/85
CPCC07K14/47C12N15/113C12N15/85C12N2310/14
Inventor 乔木武华玉吴俊静彭先文梅书棋刘贵生周佳伟孙华宋忠旭胡华李良华董斌科赵海忠
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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