Ush1c gene enhancer and its application

Abstract

The present invention discloses an Ush1c gene enhancer and its application, belonging to the field of genetic engineering, and the nucleotide sequence of the enhancer is shown in SEQ ID No. 1. The mouse embryo microinjection system is used to confirm that the sequence is an enhancer, which can significantly enhance the transcription activity of the EGFP protein gene in the inner ear. Compared with other sequences of similar size, the enhancer sequence fragment can drive the reporter gene or Ush1c to express significantly in the inner ear, and specifically express it in inner and outer hair cells, and is suitable for recombinant DNA, vectors or adenovirus and other biological materials, and is used to promote the transcription of EGFP gene or other genes in the inner ear.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to a Ush1c gene enhancer and an application thereof. Background Art

[0002] The USH1C gene is located on human chromosome 11p15.1 and encodes a scaffold protein harmonin, which contains multiple PDZ domains and plays a role in the assembly of the Usher protein complex. It is essential for the development of cochlear hair cells and hearing. Mutations in the USH1C gene can cause severe clinical symptoms in patients, including non-syndromic deafness (DFNB18, MIM: 602092) and Usher syndrome (MIM: 276904). To date, more than 70 deafness-causing variants of USH1C have been reported, and most USH1C genetic mutations that cause hearing loss follow an autosomal recessive inheritance pattern. This inheritance pattern can restore hearing by restoring the normal function of the gene through gene replacement or gene repair. However, non-specific expression of the normal USH1C gene may be harmful to the host. The USH1C gene is highly conserved in mammals.

[0003] Enhancers are typical cis-regulatory elements in the genome of eukaryotic organisms and play an important role in the regulation of gene expression. Enhancers are tissue-specific and can regulate gene expression by interacting with their target gene promoters. This interaction may be cis-acting or trans-acting. Cis-acting refers to the fact that the enhancer and its action site gene are on the same chromosome, while trans-acting refers to the fact that the enhancer and its action site gene are on different chromosomes. Enhancers contain a variety of genetic marker sites, the most common of which are transcription factor binding sites. Transcription factors, cofactors of transcription complexes, and chromatin complexes act on promoters to activate or enhance gene transcription.

[0004] Enhancers can be used to increase the specific expression of genes. It has been reported that adding enhancers to viral vectors (such as adeno-associated virus (AAV) vectors) can increase the expression activity and specificity of genes, increase transgene expression, and reduce viral doses. There are also reports that by combining different liver-specific promoters and enhancer elements to produce smaller promoter and enhancer sequences, efficient packaging and transduction of rAAV can be achieved. The expression level of rAAV containing these cis-regulatory elements and liver-specific promoters in mice is about 10 to 100 times higher than that of rAAV with a single promoter. In general, enhancers, as cis-regulatory elements, precisely regulate the expression of genes at specific times and spaces through interactions with transcription factors and chromatin remodeling complexes.

[0005] In summary, the Ush1c gene is a deafness-causing gene, and the inner ear Ush1c enhancer can be used to regulate the activation of the gene in specific developmental stages or cell types to ensure that the Ush1c gene is expressed at the right time and place. However, there has been no research and application of the Ush1c gene enhancer. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a Ush1c gene enhancer.

[0007] The technical solution of the present invention is: Ush1c gene enhancer, whose nucleotide sequence is shown in 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 Ush1c 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 specific expression of inner and outer hair cell genes.

[0012] Furthermore, the gene is Ush1c gene or EGFP gene.

[0013] The use of the enhancer or biomaterial described above in the preparation of products for enhancing the specific expression of inner and outer hair cell genes.

[0014] Furthermore, the gene is Ush1c 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 Ush1c in the inner ear, and specifically express it in inner and outer hair cells, 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 Ush1c 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 Ush1c gene enhancer-EGFP fluorescence signal in the mouse cochlea, (top: the whole inner ear cochlear basement membrane; bottom: enlarged view). 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 Ush1c 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 Ush1c gene enhancer sequence (shown in SEQ ID No. 1, with a sequence length of 691 bp) was amplified.

[0023] The primer pairs used for PCR amplification of the Ush1c gene enhancer region are as follows:

[0024] Ush1c-F:5'-CGCGGATCCGGAGTTCCCTGTATTGTTACT-3'(SEQ ID No.2)

[0025] Ush1c-R: 5'-CCGCTCGAGACACTTTCGTGTCTGAGCA-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 Ush1c enhancer can enhance the specific expression of the EGFP gene in inner and outer hair cells ( Figure 2 Middle B)

[0032] Example 2 Activity Analysis of Vectors without Ush1c Gene Enhancer Sequence

[0033] The vector without the Ush1c 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 Ush1c gene enhancer sequence has no EGFP expression (no fluorescent signal) in the inner and outer hair cells ( Figure 2 (A).

Claims

1. Ush1c 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 Ush1c 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 specific expression of inner and outer hair cell genes.

6. The use according to claim 5, characterized in that: The gene is Ush1c gene or EGFP gene.

7. Use of the enhancer according to claim 1 or the biomaterial according to claim 4 in preparing a product for enhancing the specific expression of inner and outer hair cell genes.

8. The use according to claim 5, characterized in that: The gene is Ush1c gene or EGFP gene.

Images