Application of rtl1 gene in regulation of myoblast proliferation and differentiation
A mouse myoblast, 1.RTL1 technology, applied in the field of genetic engineering
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Embodiment 1
[0026] Example 1 Changes in the expression level of RTL1 gene before and after differentiation of mouse myoblast C2C12
[0027] 1. Using fluorescent quantitative PCR to detect the expression of differentiation-related marker genes (MyoG and MyHC) and RTL1 gene mRNA levels in different differentiation stages of C2C12 cells
[0028] Undifferentiated C2C12 cells were cultured in high glucose DMEM medium containing 10% FBS. When the cells grew to 60%–70% confluence, they were replaced with 2% horse blood DMEM medium to induce myogenic differentiation. Cells were collected at 0d, 2d, and 6d of induced differentiation, respectively. The total RNA was extracted by Trizo1, reverse-transcribed with TaKaRa Primer Script RT Reverse Transcription Kit (see the instructions of the kit for the specific operation process), using SYBR Green fluorescent dye from Bio-Rad Company, LightCycler 480 fluorescent quantitative PCR instrument from Roche Company, Using β-actin as an internal reference,...
Embodiment 2
[0042] Example 2 The overexpression vector pcDNA3.1-RTL1 promotes the proliferation and differentiation of mouse myoblast C2C12 cells after overexpressing RTL1
[0043] 1. Construction of mouse RTL1 gene overexpression vector
[0044] The construction of the mouse RTL1 gene overexpression vector includes the following steps: according to the mouse RTL1 gene nucleotide sequence and the enzyme cutting site on the pcDNA3.1 vector, using the principle of homologous recombination, designing a homologous recombination arm, terminal With primers of EcoRV and XbaI restriction endonuclease sites respectively, PCR amplification obtains the complete coding region sequence of the mouse RTL1 gene, which is constructed between the EcoRV and XbaI restriction endonuclease sites of the pcDNA3.1 vector, The mouse RTL1 gene overexpression vector pcDNA3.1-RTL1 was obtained.
[0045] 1. Obtaining the coding region sequence of mouse RTL1 gene
[0046] Total RNA was extracted from mouse myoblasts ...
Embodiment 3
[0078] Example 3 siRNA inhibits the expression of RTL1 and inhibits the proliferation and differentiation of mouse myoblast C2C12 cells
[0079] 1. Design and synthesis of siRNA
[0080]According to the mouse RTL1 gene sequence (Gene ID: 353326) in Genbank, using the BLOCK-iT RNAiDesigner software (http: / / rnaidesigner.thermofisher.com / rnaiexpress / sort.do), three siRNAs were designed for different target sites of the RTL1 gene ( siRNA-1, siRNA-2, siRNA-3), and design siRNA negative control (NC), the sequence was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.
[0081] RTL1-siRNA1, sense strand: 5′-GGAGGAAUCAAGUGAUGAU-3′ (SEQ ID NO.7), antisense strand: 5′-AUCAUCACUUGAUUCCUCC-3′ (SEQ ID NO.8);
[0082] RTL1-siRNA2, sense strand: 5′-GCAUCGCACUAGAGACAUA-3′ (SEQ ID NO.9), antisense strand: 5′-UAUGUCUCUAGUGCGAUGC-3′ (SEQ ID NO.10);
[0083] RTL1-siRNA3, sense strand: 5'-GCCCAAGAACUCUCCAGAA-3' (SEQ ID NO.11), antisense strand: 5'-UUCUGGAGAGUUCUUGGGC-3' (SEQ ID NO.12).
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