CN101492673B - Africa xenopus XPAPC gene promotor and tissue specificity enhancer - Google Patents

Abstract

The invention belongs to the field of molecular biology and discloses an XPAPC gene promoter of Xenopus leavis. The promotor has a nucleic acid sequence or a sequence segment shown in SEQ ID NO:1. The invention also discloses an expression carrier containing the promoter. The invention also discloses a method of using the promoter to express a target gene. The promoter comprises a control element needed to guide the special expression of XPAPC from the early stage to the tail-bud stage of archenteron; therefore, the promoter can be used for researching the expression control situation of the specific gene (like XPAPC) during the fetation and the expression of other factors on the specific gene.

Description

XPAPC gene promoter and its tissue-specific enhancer in Xenopus laevis

technical field

The invention belongs to the field of molecular biology, and more specifically, the invention relates to Xenopus Paraaxial Protocadherin (XPAPC) gene promoter of Xenopus laevis Xenopus laevis and its application.

Background technique

Gastrulation is one of the most basic morphogenetic events in the early development of cellular organisms. Through finely regulated large-scale cell migration and rearrangement during gastrulation, the body axis of the embryo is established and the boundaries of the three forthcoming germ layers are defined. Xenopus laevis has been widely used as a representative amphibian to study cell motility and molecular mechanisms during gastrulation. Cell migration during gastrulation in Xenopus embryos has been observed and described in detail using various microscopic imaging techniques. However, little is known about the power sources of these movements and the molecular mechanisms that regulate them.

In Xenopus embryos, structural molecules including protocadherins, integrins, and fibronectins are involved in cell migration during gastrulation. The Xenopus Paraaxial Protocadherin (XPAPC) gene of Xenopus laevis is a protocadherin gene, which is first expressed in the Spemann organizer (Spemann organizer) and then in the paraaxial mesoderm in embryos undergoing gastrulation. Previous studies have shown that XPAPC can promote convergent extension (CE) during gastrulation. In addition, XPAPC is also involved in tissue separation between mesoderm and endoderm, somitogenesis and organogenesis. (organogenesis). Similar to other adhesion molecules, XPAPC participates in biological events as a member of signaling pathways as well as cellular structural molecules. Recent research work reveals that XPAPC regulates cell adhesion and migration by interacting with non-canonical Wnt signaling pathways and down-regulating the adhesion ability of intracellular C-cadherin molecules. In addition, Chung et al. recently found that there is a biologically meaningful interaction between XPAPC and ANR5, a downstream gene of the FGF signaling pathway, in gastrulation. However, in addition to Wnt5a and Lim1, which have been confirmed to activate the expression of XPAPC, the expression regulation mechanism of XPAPC is still a question to be studied.

Contents of the invention

The object of the present invention is to provide the promoter of Xenopus laevis adaxial proto-cadherin gene (XPAPC) and its application.

Another object of the present invention is to provide expression vectors and host cells containing the promoter.

In a first aspect of the present invention, an isolated nucleic acid (promoter) sequence is provided, the nucleic acid sequence being selected from the group consisting of:

(1) have the nucleic acid sequence shown in SEQID NO: 1;

(2) Nucleic acid sequence having the sequence shown in (50±49)-(4724±49) in SEQ ID NO: 1 (preferably by SEQ ID NO: 1 (50±49)-(4724±49) ) is a nucleic acid sequence consisting of the sequence shown); or

(3) A nucleic acid sequence that is more than 95% (preferably more than 99%) identical to the nucleic acid sequence defined in (1) or (2) and has the function of directing the expression of the target gene.

In another preferred example, in item (2), there is a nucleic acid sequence shown in the sequence (20±19)-(4754±19) in SEQ ID NO:1. More preferably, it is a nucleic acid sequence consisting of the sequence shown in positions (20±19)-(4754±19) of SEQ ID NO:1.

In another preferred example, in item (2), there is a nucleic acid sequence shown in the sequence (10±9)-(4764±9) in SEQ ID NO:1. Preferably, it is a nucleic acid sequence consisting of the sequence shown in positions (10±9)-(4764±9) in SEQ ID NO:1.

In the second aspect of the present invention, a vector is provided, which contains the nucleic acid sequence as a promoter element.

In another preferred example, the vector further contains a target gene sequence operably linked to the nucleic acid sequence.

In another preferred example, the target gene is a foreign gene.

In another preferred example, the target gene sequence is located downstream of the nucleic acid sequence and is a gene-coding sequence directly adjacent to the nucleic acid sequence.

In another preferred example, the distance between the nucleic acid sequence and the target gene sequence is 0-100 bp (preferably, 0-50 bp; more preferably, 0-20 bp).

In another preferred example, the target genes include (but are not limited to): Paraaxial Protocadherin (PAPC), luciferase gene, and fluorescent protein gene.

In another preferred example, the paraaxial protocadherin gene (Paraxial Protocadherin (PAPC)) is Xenopus Paraaxial Protocadherin (XPAPC) gene.

In the third aspect of the present invention, there is provided a cell (preferably a genetically engineered cell), which contains the vector.

In the fourth aspect of the present invention, the use of the nucleic acid sequence is provided, and the nucleic acid sequence is used as a promoter element to direct the expression of a target gene.

In another preferred example, the nucleic acid is used to guide the expression of the target gene according to the in vivo expression method of the paraaxial proto-cadherin gene.

In another preferred example, the target gene includes (but not limited to): Paraaxial Protocadherin (PAPC), luciferase gene, and fluorescent protein gene.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1 shows the molecular cloning and transcriptional activity analysis of the DNA sequence upstream of XPAPC.

(A) Luciferase activity of different XPAPC promoter deletion constructs.

(B) Detection of the presence of introns in the XPAPC genome sequence.

Figure 2 shows that promoter-driven expression of GFP mRNA in transgenic embryos recapitulates expression of endogenous XPAPC in spatial and temporal specificity.

(A-E) Expression of endogenous XPAPC mRNA at various stages (St).

(F-J) Expression of pFL GFP in contemporaneous transgenic embryos.

Among them, (A-B and F-G) are facing upwards, viewed from the plant pole;

Among them, (C and H) the front of the axon embryo is up, and the back view;

Among them, (D and I) the front of the neuroaxon embryo is on the left, side view;

Among them, (E and J) Lateral view of a tadpole with the front on the left.

Detailed ways

Through extensive and in-depth research, the present inventors isolated a nucleic acid from the Xenopus genome for the first time, which is located at the upstream of the Xenopus paraxial proto-cadherin gene (XPAPC gene), and it can be used as a promoter element. Direct the expression of target genes (such as reporter genes). The nucleic acid includes the regulatory elements needed to guide the specific expression of XPAPC from the early gastrulation to the tail bud stage, so that it can be used to study the expression regulation of specific genes (such as XPAPC) during embryonic development and the effects of other factors on specific genes Express. The present invention has been accomplished on this basis.

As used herein, the "promoter" or "promoter region (domain)" refers to a nucleic acid sequence, which usually exists upstream (5' end) of the coding sequence of the gene of interest, and can guide the transcription of the nucleic acid sequence into mRNA . Generally, a promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription. Herein, the promoter or promoter region includes variants of the promoter, which are obtained by inserting or deleting regulatory regions, performing random or site-directed mutations, and the like.

As used herein, "isolated" means that a substance is separated from its original environment (if the substance is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, the "operably linked" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed at a specific position relative to the nucleic acid sequence of the target gene, so that the transcription of the nucleic acid sequence is guided by the promoter region, thus, the promoter region is "operably linked" to the nucleic acid sequence.

Promoters and the gene expression they direct

In the course of the research, the present inventor cloned for the first time a nucleic acid (promoter) that can well guide the expression of exogenous genes from the upstream of the XPAPC gene, and uses the promoter to guide the expression of the reporter gene, which can well simulate the internal expression of XPAPC. source expression. In the embodiment of the present invention, the inventors have proved that the expression of GFP guided by the promoter can completely simulate the expression of endogenous XPAPC, indicating that this fragment contains the specific expression of XPAPC from the early gastrula to the tail bud stage. All the required regulatory elements, thereby changing the current situation in the prior art that the nucleic acid sequence that specifically regulates the XPAPC gene in the body is unknown.

Therefore, the present invention provides a kind of isolated nucleic acid (promoter), described nucleic acid has: the sequence shown in SEQ ID NO: 1, or has sequence (50 ± 49)-(4724 ± 49 in SEQ ID NO: 1 ), the nucleic acid can be used as a promoter element to guide the expression of the target gene.

In addition, the present invention also includes some variants of the above nucleic acids with the same function. Including: the sequence has more than 95% (preferably 99%) homology with SEQ ID NO: 1 or the sequence shown in (50±49)-(4724±49) in SEQ ID NO: 1 and has a guiding target gene Nucleic acid expressing function.

The promoter of the present invention can be operably linked to a gene of interest, which can be foreign (heterologous) with respect to the promoter. The target gene can generally be any nucleic acid sequence (such as a structural nucleic acid sequence); the target gene preferably encodes a protein with specific functions, such as some proteins with important properties or functions; or the target gene is A gene that can function as an indicator after transcription or expression, such as a reporter gene.

For example, the target gene includes but not limited to: Xenopus laevis paraaxial proto-cadherin gene, luciferase gene, green fluorescent protein gene.

The promoter of the present invention can also be operably linked to an improved gene sequence of interest which is foreign (heterologous) to the promoter. The gene of interest can be modified to produce various desired properties. For example, the target gene can be improved to increase the content of essential amino acids, improve the translation of amino acid sequences, change post-translational modifications (such as phosphorylation sites), transport translation products outside the cell, improve protein stability, insertion or deletion cell signaling, etc.

Additionally, promoters and genes of interest can be engineered to downregulate specific genes. This is generally accomplished by linking the promoter to the gene sequence of interest directed in antisense reverse. Those of ordinary skill in the art are familiar with such antisense technology. Any nucleic acid sequence can be modulated in this manner.

The promoter of the present invention can also be used to guide the expression of exogenous genes, study the functions of important molecules in the development of mesoderm and inner ear development, establish animal models of developmental defects, and gene therapy for developmental defects.

The promoter and target gene sequence of the present invention can be contained in a recombinant vector.

The recombinant vector generally includes (from 5' to 3' direction): a promoter that guides the transcription of the target gene, and the target gene. If necessary, the recombinant vector may also include a 3' transcription terminator, a 3' polynucleotide signal, other non-translated nucleic acid sequences, transport and targeting nucleic acid sequences, resistance selectable markers, enhancers or operators.

Methods for preparing recombinant vectors are well known to those skilled in the art. The term "expression vector" refers to bacterial plasmid, bacteriophage, yeast plasmid, mammalian cell virus or other vectors well known in the art. In conclusion, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. Preferably, the expression vector is a eukaryotic expression vector.

Methods well known to those skilled in the art can be used to construct an expression vector containing the promoter and/or target gene sequence of the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes, such as dihydrofolate reductase, neomycin resistance, hygromycotic Antibiotic resistance, etc.

In addition to the promoter of the present invention, the recombinant vector may also contain one or more other promoters. Said other promoters are, for example: tissue-specific, constitutive or inducible.

As an example of the present invention, the vector is pGL3-Basic, which itself contains the sequence encoding the luciferase gene. Through transformation, that is, using the multiple cloning site on pGL3-Basic, the promoter region of the present invention can be constructed in front of the luciferase coding gene, and transformed into host cells, the promoter will activate the expression of the luciferase coding gene, so The above-mentioned initiation is regulated by various cis-acting elements in the promoter region, simulating the situation that the gene is activated and transcribed in vivo.

The vector containing the above-mentioned appropriate promoter and target gene can be used to transform appropriate host cells, or transformed into fertilized eggs or embryos, so that they can express proteins.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples include Escherichia coli, yeast, animal tissue cells, and the like. The fertilized eggs or embryos may be amphibian fertilized eggs or embryos, for example, Xenopus laevis fertilized eggs or embryos.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription.

Those of ordinary skill in the art will know how to choose an appropriate vector, promoter, enhancer or host cell.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

Methods for transforming fertilized eggs or embryos with recombinant DNA are also known in the art, and microinjection techniques are usually used. For example, the transgenic operation can be performed according to the method described in Kroll KL et al. (1996). Transgenic Xenopus embryos fromsperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122: 3173-3183. Plasmids used as transgenes can be linearized in advance.

In the example of the present invention, under the guidance of the promoter, the luciferase (Luciferase) gene and the green fluorescent protein (GFP) gene can be well expressed. Therefore, it can be seen that the promoter of the present invention has important application value in the study of gene expression regulation.

In the example of the present invention, after replacing the coding sequence of luciferase in the full-length XPAPC promoter luciferase analysis plasmid with the coding sequence of green fluorescent protein, a reporter plasmid for preparing transgenic Xenopus embryos was obtained. The present inventors prepared transgenic Xenopus embryos at different stages and performed in situ hybridization experiments with probes reflecting GFP expression. The results showed that, in the transgenic Xenopus embryos at different stages, the expression pattern of GFP directed by the XPAPC promoter was very similar to that of the endogenous XPAPC expression pattern, which indicated that the XPAPC promoter obtained by the inventor contained the known XPAPC in Xenopus All important cis-acting elements required for early embryonic expression.

The main advantages of the present invention are:

A promoter that can guide the expression of the target gene is disclosed, and the expression regulation of specific genes (such as XPAPC) and the expression of other factors (such as some signaling pathways) to specific genes can be studied during embryonic development by using the promoter of the present invention.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described in the manufacture conditions recommended by the manufacturer.

Embodiment 1 Embryo culture and transgenic method

Xenopus embryo culture operation: fertilized eggs were cultured as described in Fang PF et al. (2004), Multiplesignaling pathways control Tbx6 expression during Xenopus myogenesis.Acta Biochim Biophys Sin (Shanghai) 36: 390-396, staging according to Nieuwkoop and Faber (Normal Table of Xenopus Laevis, Garland Publishing, 1994).

Preparation of transgenic embryos of Xenopus laevis: According to the method described in Kroll KL et al. (1996). Plasmids used as transgenes were digested with Smal to linearize. Each plasmid was subjected to 3 independent rounds of transgenic manipulation.

Cloning of embodiment 2 XPAPC 5' upstream control sequence

Since the genome sequence of Xenopus laevis has not been sequenced, the inventor cloned the upstream regulatory sequence of XPAPC through a unique design, as follows:

Genomic DNA isolation : take 2g fresh liver tissue of adult male Xenopus laevis, put it into a mortar and add liquid nitrogen to grind thoroughly, extract twice with phenol/chloroform method, take the supernatant and precipitate with ethanol, wash once with 75% ethanol, dry and dissolve In deionized water, quantified by a spectrophotometer.

Ligation : 1 μg of genomic DNA was digested overnight with 10 U of EcoR I, and after digestion, 1 μg of genomic DNA was ligated with 50 mM PCR linker at 16°C with T4 DNA ligase overnight, recovered by ethanol precipitation, and dissolved in 20 μl of deionized water.

The primers used in PCR are as follows:

AP1 (SEQ ID NO: 2): 5'-GTAATACGACTCACTATAGGC-3';

AP2 (SEQ ID NO: 3): 5'-ACAATAGGGCACGCGTGGT-3';

GSP1 (SEQ ID NO: 4): 5'-CAACACTGCAATTACAGTGCCAGGGGGTTTCTTCTTC-3';

GSP2 (SEQ ID NO: 5): 5'-GAGAAGCAGCATCTTGAAGAATCAAAGTTGCACC-3'.

Primers for AP1 and AP2 are based on the Genome Walker adapter system.

Two rounds of adapter PCR (Adaptor PCR) to separate and increase the 5'upstream sequence of XPAPC :

In the first round, 50 μl system was used, and the dosage was as follows:

10× buffer 5μl;

dNTP 250 μl;

GSP1 10pM;

AP1 10pM;

100ng of digested genomic DNA with adapters connected;

LA Taq polymerase 2.5U;

Make up to 50 μl with deionized water.

PCR program:

Denaturation at 94°C for 3 minutes;

The first step is 10 cycles: denaturation at 94°C for 30 seconds;

Annealing at 70°C for 30 seconds;

Extend at 72°C for 5 minutes;

The second step is 30 cycles: denaturation at 94°C for 30 seconds;

Annealing at 60°C for 1 minute;

Extend at 72°C for 5 minutes;

Finally extend at 72°C for 10 minutes.

The second round of PCR: Dilute the first round of PCR products 100 times and add samples according to the following proportions, and the dosage is as follows:

10 × buffer (plus Mg ²⁺ ) 5μl;

dNTP 250μl;

GSP2 10pM;

AP2 10pM;

The first round of PCR product was diluted 100 times to 100ng;

LA Taq polymerase 2.5U;

Make up to 50 μl with deionized water.

PCR was performed using the same procedure as the first round.

The product of the second round of PCR was separated with 1% agarose gel, and a single band at 4.3 kb was identified, recovered by tapping the gel, loaded into a T-easy vector (Promega), and sequenced.

As a result, a DNA fragment with a length of 4914bp was cloned, as shown in SEQ ID NO:4. It overlaps with the 5' region of the published Xenopus PAPC gene (see Kim SH et al. (1998), The role of paraaxial protocadherin in selective adhesion and cell movements of themesoderm during Xenopus gastrulation. Development 125: 4681-4690).

Embodiment 3 Contains the construction of the vector of promoter and reporter gene

After the PCR amplified fragments obtained above are purified by conventional methods, the fragments of sequences 1-4773 in SEQ ID NO: 1 and carrying XhoI/HindIII restriction sites at both ends are obtained by conventional PCR methods, and inserted into PGL3 digested by XhoI/HindIII - In Basic (Promega, with its own luciferase encoding gene), the p-4773Luc construct was obtained.

The present inventor also utilizes XbaI/HindIII digestion p-4773Luc, the luciferase gene wherein is removed, reclaims the plasmid through processing; The plasmid (obtained from Ralph Rupp's laboratory) was excised with XbaI and HindIII) and loaded into the previously processed vector to obtain the plasmid p-4773GFP used for transgenesis.

The present inventor also constructed a fragment consisting of the 1-2920th sequence in SEQ ID NO: 1, and loaded the sequence into PGL3-Basic and green fluorescent protein (GFP) reporter plasmids using the same method as described above, thereby obtaining Plasmids p-2920Luc and p-2920GFP were tested.

The luciferase reporter plasmid constructed above will be used to determine the activity of the reporter gene, and the green fluorescent protein reporter plasmid will be used to conduct Xenopus transgenic embryo experiments.

Transcription regulation analysis of embodiment 4 promoter

For luciferase assays, 2 nl solutions (solvent: DEPC water) containing 25 pg of different luciferase test plasmids and 25 pg of the internal standard reporter gene pRL-SV40 (purchased from Promega) were injected into animal poles of four-cell stage embryos.

In order to analyze whether the obtained XPAPC 5' upstream sequence has transcriptional activity, the previously constructed p-4773Luc, p-2920Luc vector, PGL3-Basic empty vector (each 25pg, 2nl) and internal reference control pRL-SV40 (25pg, 2nl) Inject into four-cell-stage Xenopus embryo animal poles respectively, isolate animal pole caps at around stage 8.5, and then culture to stage 12.5 to detect the luciferase activity of the reporter gene. Luciferase assays were performed with the Dual-Luciferase Reporter Assay System (Promega).

The results are shown in Figure 1A, showing that the 5' upstream sequence fragment of XPAPC has transcriptional activity.

In eukaryotes, introns, especially the first intron, may also be involved in the regulation of gene expression. In order to detect the presence or absence of introns in the genomic sequence of XPAPC, the present inventors performed genomic PCR.

The results are shown in Figure 1B, after adding primers (upstream: TTG TTT TAA ATG ACT GCAGGT CTG GAA GGA (SEQ ID NO: 6); downstream : In the PCR reaction of CAG TTC CCA TTT TCT GGACCC TTG TTG A (SEQ ID NO: 7)), only one fragment with a length of 3.6 kb can be amplified. DNA sequencing of this fragment revealed that the XPAPC genome sequence does not contain introns like some other proto-cadherins.

In order to detect whether the obtained XPAPC 5' upstream regulatory sequence can guide the expression of the reporter gene GFP in Xenopus embryos, the inventors used SmaI to linearize the plasmid p-4773GFP (pFL-GFP) containing the upstream regulatory sequence, and transfected Introduced into Xenopus embryos, and then detected the expression of reporter gene GFP during embryonic development by in situ hybridization, and detected the expression of endogenous XPAPC in untransformed embryos. The transcripts of XPAPC and GFP were detected by whole-mount in situ hybridization with XPAPC or GFP probes, respectively.

The preparation of the mRNA template was described in the literature Fang PF et al. (2004), Multiple signaling pathways control Tbx6 expression during Xenopus myogenesis. ActaBiochim Biophys Sin (Shanghai) 36: 390-396. In situ hybridization of embryos using Digoxigenin (DIG)-labeled XPAPC or GFP probes (XPAPC probes refer to Kim SH et al., The role of paraaxial protocadherin in selective adhesion and cell movements of the mesoderm during Xenopus gastrulation. Development 125: 4681- described in 4690.; the GFP probe is obtained by in vitro transcription with T7 RNA polymerase after linearizing pCS2-GFP), according to Harland RM. 1991.In situ hybridization: an improved whole-mount method for Xenopus embryos.Methods Cell Performed as described in Biol 36:685-695. Hybridized embryos were treated with bleach solution (0.5×SSC containing 1% hydrogen peroxide and 5% formamide) under fluorescent light.

It was found that in Xenopus embryos, XPAPC was expressed as early as the late blastocyst stage (Stage 9.5 (St9.5)), one and a half hours before the start of gastrulation. At this stage, XPAPC expression could be detected in the dorsal marginal zone using whole-mount embryo in situ hybridization (Fig. 2A). In the early stage of gastrula (stage 10), the expression of XPAPC extended to the entire marginal zone and showed a pattern of more dorsal and less ventral (Fig. 2B). As gastrulation progressed, the expression of XPAPC in the dorsal midline was downregulated as the axial mesoderm (notochord) began to develop. After the mesoderm cortex was fully involuted at stage 13, XPAPC mRNA expression anteriorly had a clear border separating the head and trunk mesoderm (Fig. 2C). Once the somitogenesis begins, the expression of XPAPC will be down-regulated in the mature somites and the banded expression will move to the rear of the embryo (Fig. 2D). Expression was maintained on the tip of the tail (stage 24) (Fig. 2E). In addition to the expression in the mesoderm, from the 17th stage, XPAPC is also expressed at the position where the cochlea is about to be formed on both sides of the head (Fig. 2D). Later, with the development and involution of the cochlea, the expression of XPAPC is also more limited to the cochlea When the ear cup is formed, XPAPC is expressed inside the cup-like structure (Fig. 2E).

In Xenopus embryos that have undergone transgenic manipulations to integrate the linearized pFL-GFP reporter plasmid into the genome, the expression pattern of GFP in both gastrulation mesoderm and developing cochlea is similar to that of endogenous The expression of XPAPC was very similar (Figure 2F to J). It shows that the promoter having the sequence of positions 1-4773 in SEQ ID NO: 1 contains the regulatory elements needed to guide the specific expression of XPAPC from the early gastrula to the tail bud stage.

In addition, the present inventors used a similar method to use the p-2920GFP plasmid to verify that the promoter of the 1-2920th sequence in SEQ ID NO: 1 can guide the expression of GFP, but the expression is different from that of endogenous XPAPC, which is due to Caused by the absence of certain regulatory elements in this sequence.

In addition, the present inventors have verified that the nucleic acid consisting of the sequence 3-4769 in SEQ ID NO: 1 also has an activity similar to the sequence 1-4773 by a similar method.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

<120> XPAPC gene promoter and its tissue-specific enhancer in Xenopus laevis

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<170>PatentIn version 3.3

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tccagatagt tccgattagtac aggaaggcca tgttccatatag actccatttt atctaaataa 2220

tccagatttt tatcaactat ttcctttatc tctgtaaggt tacagacaca cgctaagatt 2280

caggggagatt tagtcacctc ttcttcggac gactaatctc cccgaaaagc cttcctgccg 2340

gctagaatct gaatcgctgg caggatgata agcggaccaa cttgttttca gaagtcgccc 2400

aacgttgcct cacaaggaga ctttgggcaa ttttggaaaa caaatcgttc cgagtgccac 2460

tctgccggtg atttagattt tagccggcgg aaaggctttt cggggagatt agtcgccgga 2520

agaagaggcg ataaatctcc ccgaatctta gcgtgtgctc ttacccctaat aataaaatag 2580

tagcttgtac ttgatccaaa ctaagatata attaatcctt attgaaggca aatccaagct 2640

attgggttta ttaaatgttt acatgatttt ctagtatact taaggtatga agatccaaat 2700

tacggaaaaa tcagttaccc agaaaaccccc aggtcccgag cattatgtat agcattctat 2760

atagcaggaa tattgcatac ctgcatatac aacttaaacc catcatataa aaataaaaaa 2820

tgtgtatctc cttgtgtatc ctttcattag atggagtgtg aattgtaatt aaccccttaa 2880

ctcttggcat gtttttctac aggaaacaaa ctgcaatacg atcatataac acaggcagcc 2940

tcaccacatc ctaccttcaa agctcacagg atatgttaat gtccatgtca ttggcaccaa 3000

aggatttatg ttgagcagag cagagacacc attgtctctt cttaaataaa cactattagc 3060

tgtggaatga aattagcaca ggataaattg ctctttaatt aaaaatttca attactataa 3120

atcaacatac tgcagtgggg ctaaagttat acactgtacc ctgcccctga aagtcagact 3180

actgacatat acatatttatttttatta ttatatttat ctctatctta cttaaattta 3240

ggtgcacatt tattcttgct tttttggcac aggtaacaaa catttctaaa gttactaatc 3300

atttttgtac attgctaccc atttacccaa cgacaattat tgctggttca gtaagtaaga 3360

taagacttac attcaaagca tataagatta gttaaccaga aacccattaa ccagaaagtt 3420

cagaattaca ggaaggccat ctcccataga gtctatttta ataaaataat gtacattttt 3480

aaaaatgatt tccttttttt ctattataat aacacagtag cttatacttg atccttacta 3540

agacataatt aatcctattg gtagtgatgg gagaaattgg ggcattttgc ttcgccgaaa 3600

aatgtgcgaa tttcccgcga aacggcaaaa attcgcaaaa cggcgttggc gtccattttt 3660

ggcgccagac atcaaaaaat tcgcccatca ctacctattg ggtttatta atgtttaaag 3720

gattttttag ttgaccaaat cacagaaaga cccccttatc tggaaaacca caggttcatg 3780

agcattctgg ataacaggtc ccatacctgt acagtgaaaa tatttcaggg catttgtcct 3840

gtgcctctat aatcagagga gcccagaaaa atgtacaaag gacatataac gttgcttacc 3900

tattgttaaa tattttcaat agggatgtca gcagtttaag ctcttgcaga atcgtgtcag 3960

gtgattctgg gagttgtagt tcaataacag ctagagaaca gctaattaga accccctgat 4020

accctcccaa cttcattgca ttaaaaggat cccattcttc tcttcctatg cccctgttaa 4080

ggtaaacatt tttatccaat ctagcaccat tatggggttt tatataaaga tctaccttat 4140

gaatccccat aaatatgaca actaacaaaa aataggtaca gatgactgtt tttattttga 4200

gacttatagt aaacattttt atccaatcta gcacaattat ggggttttat ttaatgatct 4260

tccttatgaa tccccataaa tatgaccagt cacaaataat gtgtatggat gtttttagtt 4320

tgagactgtt aatattatac attttagtt acaattcaag acaatgttat agactgtatg 4380

aacagagtat tatattctat caaatgtggt aaaaaatgaa aagcataata aattacatag 4440

taaaaaataa ggtatatcag aatatttctt taatttaatg aaattgtttt agtaacaaat 4500

attttgtttg ttgttcccac tcaagaatga ttctctgcat tgcgcttgct tctttatgaa 4560

acactgattt aaaatgaaac attgtcataa aatataacag tttgttggac ttccaagtga 4620

gacctatttg atgacagtct cattagctct gccccagtgc tcctttgtct ctggggaaag 4680

ggattggcta gaagtaagtt catttacata tcctctggga atataacctg ggggcagaga 4740

tggaaccaag cagacaaaca ggatcattcc tacagagatg aactccttga gattgtttta 4800

aatgactaca ggtctggaag gattcacatt gccaacactgt ttctaggcag gaaaaaactg 4860

caagtttcaa ctttgttttt ggtgcaactt tgattcttca agatgctgct tctc 4914

<210>2

<211>21

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>2

gtaatacgac tcactatagg c 21

<210>3

<211>19

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>3

acaataggggc acgcgtggt 19

<210>4

<211>36

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>4

caacactgca attacagtgc cagggggttc ttcttc 36

<210>5

<211>34

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>5

gagaagcagc atcttgaaga atcaaagttg cacc 34

<210>6

<211>30

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>6

ttgttttaaa tgactgcagg tctggaagga 30

<210>7

<211>28

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>7

cagttcccat tttctggacc cttgttga 28

Claims (9)

1. an isolating promotor is characterized in that, said promotor is selected from down group:

(1) nucleic acid shown in the SEQ ID NO:1; Or

(2) nucleic acid shown in 1-4773 position or the 3-4769 position among the SEQ ID NO:1.

2. a carrier is characterized in that, described carrier contains the described promotor of claim 1.

3. carrier as claimed in claim 2 is characterized in that described carrier also contains the target gene sequences that is operably connected with described promotor.

4. carrier as claimed in claim 3 is characterized in that, described promotor and target gene sequences be 0-100bp at interval.

5. carrier as claimed in claim 3 is characterized in that, described goal gene comprises: next to axis protocalcium Fibronectin gene, luciferase gene or fluorescence protein gene.

6. a cell is characterized in that, described cell contains the described carrier of claim 2.

7. the purposes of the described promotor of claim 1 is characterized in that, described promotor is used to instruct the expression of goal gene.

8. purposes as claimed in claim 7 is characterized in that, described promotor is used to instruct goal gene to express according to phraseology in the next to axis protocalcium Fibronectin genosome.

9. purposes as claimed in claim 7 is characterized in that, described goal gene comprises: next to axis protocalcium Fibronectin gene, luciferase gene or fluorescence protein gene.

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