Mitf gene enhancer and its application in improving gene expression specificity in cochlear stria vascular cells

Abstract

The present invention discloses a Mitf gene enhancer and its application in improving the gene-specific expression of cochlear stria vascular cells, and relates to the field of gene expression and regulation technology. The nucleotide sequence of the enhancer is shown in SEQ ID No. 1. The mouse embryo microinjection system was used to confirm that the sequence is an enhancer, which can significantly enhance the transcription activity of the EGFP protein gene in the cochlear stria vascular cells. Compared with other sequences of similar size, the enhancer sequence fragment can drive the reporter gene or Mitf to be specifically expressed in the cochlear stria vascular cells and the expression level is significantly enhanced. The enhancer is suitable for biological materials such as recombinant DNA, vectors or adenoviruses, and is used to promote the transcription of the EGFP gene or other genes in the cochlear stria vascular cells.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a Mitf gene enhancer and its application in improving the gene expression specificity of cochlear stria vascular cells. Background Art

[0002] Deafness is the most common sensory disorder and seriously affects the quality of human life. Deafness caused by genetic factors is called hereditary deafness, which is divided into syndromic deafness (SHL) and non-syndromic deafness (NSHL) according to the accompanying symptoms. Waardenburg syndrome (WS), also known as hearing-pigment syndrome, has an incidence rate of 1 / 42,000 in the population and is one of the most common syndromic deafness; about 2% to 5% of prelingual deafness is caused by WS. The main clinical manifestations of WS are sensorineural deafness, accompanied by abnormal distribution of iris, hair and skin pigments, limb malformations and congenital mega-sigmoid colon.

[0003] At present, MITF, PAX3, SOX10, EDNRB, SNAI2 and EDN3 genes are known to be pathogenic genes of WS, among which the most important is MITF gene (Microphthalmia-associated Transcription Factor). A retrospective analysis of clinical studies on molecular genetics related to WS found that the carrier rate of MITF gene mutation in WS patients is about 28%; WS patients carrying MITF gene mutations mainly show WS2 type, with a deafness rate as high as 89.6%, and 60% of them are severe deafness. WS2 type patients have sensorineural deafness and abnormal pigment distribution as the main clinical features, usually without other symptoms. The human MITF gene is located on chromosome 3p13, and its 9 exons can form multiple splice types through selective splicing; the main splice type in melanocytes is M-MITF, which consists of 419 amino acids. The MITF gene belongs to the Myc superfamily and is a transcription factor containing a basic helix-loop-helix leucine zipper (bHLH-zip) structure. Its basic structural domain bHLH-zip is highly conserved in different species, indicating that it has important biological functions in the process of evolution. It is currently believed that the bHLH-zip structure enables MITF to bind specifically to DNA, and the main function of the MITF gene is to act as a key transcription factor and general regulatory factor in regulating the development of melanocytes during the differentiation of neural crest cells into melanocytes, regulating the growth and survival of melanocytes and the synthesis, transport and transportation of melanin.

[0004] In WS2 patients, WS caused by MITF gene mutations is mainly due to haploid insufficiency. In theory, gene replacement therapy can be used to deliver normal genes to provide sufficient normal functional proteins for gene therapy. The mammalian cochlea is a complex system composed of many different types of cells, and these cell types have different functions due to their unique gene expression patterns. The MITF gene in the cochlea is mainly expressed in the stria vascularis cells, which are composed of marginal cells (MCs), intermediate cells (ICs) and basal cells (BCs). They pump potassium ions from the perilymph to the endolymph through ion channels; the endolymph potential generated by the stria vascularis is necessary for high-sensitivity hearing conduction.

[0005] At present, the main target cells for gene therapy of deafness are hair cells, and there are few studies on gene therapy targeting vascular striae cells. In order to achieve efficient transfection of vascular striae cells by AAV virus, we identified an enhancer of Mitf gene. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a Mitf gene enhancer.

[0007] The technical solution of the present invention is: Mitf gene enhancer, whose nucleotide sequence is shown as SEQ ID No.1.

[0008] The primer pair for amplifying the enhancer described above, the nucleotide sequence of the primer pair is shown as SEQ ID No.2 and SEQ ID No.3.

[0009] The method for amplifying the enhancer described above is to use mouse genomic DNA as a template and perform PCR amplification with the primer pair shown in SEQ ID No.2 and SEQ ID No.3 to obtain the enhancer of the Lhfpl5 gene shown in SEQ ID No.1.

[0010] The biological material containing the enhancer described above is recombinant DNA, vector, adenovirus or engineered bacteria.

[0011] The use of the enhancer or biomaterial described above in enhancing the gene-specific expression of cochlear stria vascular cells.

[0012] Furthermore, the gene is Mitf gene or EGFP gene.

[0013] The use of the enhancer or biomaterial described above in the preparation of a product for enhancing the gene-specific expression of cochlear stria vascular cells.

[0014] Furthermore, the gene is Mitf gene or EGFP gene.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The mouse microinjection system was used to confirm that the sequence is an enhancer, which can significantly enhance the transcriptional activity of the EGFP protein gene in the inner ear. Compared with other sequences of similar size, the enhancer sequence fragment can significantly enhance the expression of the reporter gene or Mitf gene in the inner ear, and specifically express it in the cells of the cochlear stria vascularis, and is suitable for biological materials such as recombinant DNA, vectors or adenoviruses to promote the transcription of the EGFP gene or other genes in the inner ear. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Schematic diagram of the vector structure after Mitf enhancer and EGFP were cloned into the pWHERE plasmid;

[0018] Figure 2 : Comparison of the fluorescence effect of the mouse inner ear when using different enhancers; Among them: Group A empty vector-EGFP has no fluorescence signal in the mouse cochlea; Group B Mitf gene enhancer-EGFP has a fluorescence signal in the mouse cochlea. DETAILED DESCRIPTION

[0019] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0020] Example 1 Cloning and activity analysis of Mitf gene enhancer sequence

[0021] (1) The Hsp68 promoter sequence was cloned upstream of the reporter gene EGFP, and the activity of the enhancer was detected by detecting the expression of the EGFP gene. The pWHERE vector and the gene promoter sequence were connected by Qingke Biotechnology Co., Ltd., and the successfully connected vector was named pWHERE-promoter vector.

[0022] (2) DNA was extracted from the inner ear tissue of mice and the enhancer sequence of the Mitf gene (shown in SEQ ID No. 1, with a sequence length of 626 bp) was amplified.

[0023] The primer pairs used for PCR amplification of the enhancer region of the Mitf gene are as follows:

[0024] Mitf-F:5'-CGCGGATCCAAGCTGCTGTTATTACGAC-3'(SEQ ID No.2)

[0025] Mitf-R: 5'-CCGCTCGAGCAAATGATAAACCAAGCCAG-3' (SEQ ID No. 3).

[0026] (3) Subsequently, the enhancer sequence was transferred into the pWHERE-promoter vector using the BamHI and XhoI restriction sites. The constructed plasmid is shown in Figure 1 .

[0027] (4) Prepare linearized transgenic DNA: Use appropriate restriction endonucleases BamHI and XhoI to cut the plasmid to linearize the exogenous DNA from the vector, and then purify the exogenous DNA to remove impurities and enzymes. Finally, dissolve the purified DNA in TE buffer and adjust the concentration to 1-2 ng / μL, and store at 4°C for future use.

[0028] (5) Obtaining mouse fertilized eggs: Select healthy, age-appropriate female mice (usually 3-4 weeks old) for superovulation. 12-14 hours after superovulation, mate these female mice with healthy, sexually mature male mice. The next morning, dissect the female mice and collect the fertilized eggs in their oviducts. The fertilized eggs cultured in vitro should have two obvious pronuclei (male and female) and are ready for injection.

[0029] (6) Microinjection of exogenous DNA: The collected fertilized eggs are placed in a culture medium containing HEPES buffer, and the DNA solution is directly injected into the pronucleus (usually the larger male pronucleus) using a microinjection needle. After the injection is completed, the fertilized eggs are transferred to a culture dish containing appropriate culture medium and continue to incubate. Until they are implanted into the oviduct of a pseudopregnant female mouse, usually 10-20 fertilized eggs are implanted into each female mouse. After the transplant is completed, the female mouse is raised alone and its pregnancy status is observed.

[0030] (7) Identification of transgenic mice: After successful embryo transfer, wait for the required period to take out the mouse embryo for testing. Cut off the head of the mouse embryo, open the skull, remove the brain tissue, expose the temporal bone and remove the cochlea. After the cochlea is removed, move it into a culture dish containing pre-cooled 1XPBS; under a dissecting microscope, use a sharp needle to pick up the bone wall of the volute at the apex of the cochlea, and then peel the volute along the gap between the spiral ligament and the volute until the complete spiral ligament is fully exposed; then use a sharp needle or tweezers to separate and remove the entire spiral ligament, and immediately transfer it to a 2ml centrifuge tube containing 4% paraformaldehyde and fix it at 4℃ for 30 minutes. After 30 minutes, remove the spiral ligament under a dissecting microscope, and lay the entire cochlear basement membrane flat on a slide, add DAPI and seal the slide. Finally, under a fluorescence microscope, select the DAPI fluorescence channel to observe the nuclear signal of the basement membrane, the 488nm laser excitation channel to observe the EGFP signal, and take pictures of the entire complete basement membrane, the top, middle and base of the local basement membrane.

[0031] It can be seen that the Mitf enhancer can enhance the expression of the EGFP gene specifically in the cochlear stria vascularis cells ( Figure 2 Middle B)

[0032] Example 2 Activity Analysis of Vectors without Mitf Gene Enhancer Sequence

[0033] The vector without the Mitf gene enhancer sequence was microinjected, and the other operation steps were the same as in Example 1. It can be seen that the vector without the Mitf gene enhancer sequence has no EGFP expression (no fluorescent signal) in the cochlear stria vascularis cells ( Figure 2 (A).

Claims

1. Mitf gene enhancer, the nucleotide sequence of which is shown in SEQ ID No.

1.

2. A primer pair for amplifying the enhancer according to claim 1, characterized in that: The nucleotide sequences of the primer pair are shown in SEQ ID No.2 and SEQ ID No.

3.

3. The method for amplifying the enhancer according to claim 1, characterized in that: Using mouse genomic DNA as a template, PCR amplification was performed with the primer pair shown in SEQ ID No. 2 and SEQ ID No. 3 to obtain the enhancer of the Mitf gene shown in SEQ ID No.

1.

4. A biological material containing the enhancer according to claim 1, wherein the biological material is recombinant DNA, a vector, adenovirus or an engineered bacterium.

5. Use of the enhancer according to claim 1 or the biomaterial according to claim 4 in enhancing the gene-specific expression of cochlear stria vascularis cells.

6. The use according to claim 5, characterized in that: The gene is Mitf gene or EGFP gene.

7. Use of the enhancer according to claim 1 or the biomaterial according to claim 4 in the preparation of a product for enhancing the gene-specific expression of cochlear stria vascular cells.

8. The use according to claim 5, characterized in that: The gene is Mitf gene or EGFP gene.

Images