CN101492673A - Africa xenopus XPAPC gene promotor and tissue specificity enhancer - Google Patents
Africa xenopus XPAPC gene promotor and tissue specificity enhancer Download PDFInfo
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- CN101492673A CN101492673A CNA2008100330862A CN200810033086A CN101492673A CN 101492673 A CN101492673 A CN 101492673A CN A2008100330862 A CNA2008100330862 A CN A2008100330862A CN 200810033086 A CN200810033086 A CN 200810033086A CN 101492673 A CN101492673 A CN 101492673A
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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
Technical field
The invention belongs to biology field, more specifically, the present invention relates to Africa xenopus Africa xenopus next to axis protocalcium Fibronectin gene (Xenopus Paraxial Protocadherin (XPAPC)) promotor and uses thereof.
Background technology
In the early development of cell biological, former bowel movement is one of the most basic form generation incident.By the mass cell migration and the rearrangement of finely regulating in the former bowel movement, embryo's axon is set up, and the border that is about to three germinal layers of formation is also determined.Africa xenopus Xenopus laevis is widely used motion and the molecular mechanism of studying cell in the former bowel movement as the representative of Amphibians.By using various micro-imaging techniques, the cell migration in the former bowel movement of Africa xenopus embryo is at length observed and is described.Yet, few known to also about the molecular mechanism of the power resources of these motions and these motions of regulation and control.
In the Africa xenopus embryo, the structural molecule that comprises protocalcium Fibronectin (Protocadherin), integrin (integrins) and fibronectin (fibronectin) all participates in the cell migration in the former bowel movement.Xenopus Paraxial Protocadherin (XPAPC) gene of Africa xenopus is a protocalcium Fibronectin gene, it at first is expressed in Spemann organizer (Spemann organizer) in the embryo who carries out former bowel movement, be expressed in paraxial mesoderm subsequently.The previous XPAPC that studies show that can promote to converge stretching (convergent extension in former bowel movement, CE), in addition, XPAPC also participates in separate tissue (tissue separation), body segment formation (somitogenesis) and the organ generation (organogenesis) between mesoderm and the entoderm.Similar with other adhesion molecule, XPAPC as the cellularstructure molecule time also the member as signal path participate in the biology incident.Nearest research work disclose XPAPC by interact with non-classical Wnt signal path and the downward modulation cell in the adhesive capacity and the migration that come regulating cell of the adhesive capacity of C-cadherin molecule.In addition, there is the interaction with biological significance in the downstream gene ANR5 of people's recent findings XPAPC such as Chung and FGF signal path in former bowel movement.Yet except Wnt5a and Lim1 are studied the expression that confirmation can activate XPAPC, remain a problem that requires study about the expression and regulation mechanism of XPAPC.
Summary of the invention
The object of the present invention is to provide promotor of the paraxial protocalcium Fibronectin of Africa xenopus gene (XPAPC) and uses thereof.
Another object of the present invention is to provide the expression vector and the host cell that contain described promotor.
In a first aspect of the present invention, a kind of isolating nucleic acid (promotor) sequence is provided, described nucleotide sequence is selected from down group:
(1) has the nucleotide sequence shown in the SEQ ID NO:1;
(2) has the nucleotide sequence of sequence shown in the position, (50 ± 49)-(4724 ± 49) among the SEQ ID NO:1 (being preferably the nucleotide sequence of forming by sequence shown in the position, SEQ ID NO:1 (50 ± 49)-(4724 ± 49)); Or
(3) nucleotide sequence that limits with (1) or (2) has more than 95% (preferred more than 99%) homogeny and has the nucleotide sequence that instructs the destination gene expression function.
In another preference, in (2) item, has the nucleotide sequence of sequence shown in the position, (20 ± 19)-(4754 ± 19) among the SEQ ID NO:1.Preferred, by the nucleotide sequence that sequence is formed shown in the position, (20 ± 19)-(4754 ± 19) among the SEQ ID NO:1.
In another preference, in (2) item, has the nucleotide sequence of sequence shown in the position, (10 ± 9)-(4764 ± 9) among the SEQ ID NO:1.Be preferably nucleotide sequence by sequence is formed shown in the position, (10 ± 9)-(4764 ± 9) among the SEQ ID NO:1.
In a second aspect of the present invention, a kind of carrier is provided, described carrier contains described nucleotide sequence, as promoter element.
In another preference, described carrier also contains the target gene sequences that is operably connected with described nucleotide sequence.
In another preference, described goal gene is a foreign gene.
In another preference, described target gene sequences is positioned at the downstream of described nucleotide sequence, and is and the direct sequence of contiguous encoding gene of described nucleotide sequence.
In another preference, described nucleotide sequence and target gene sequences be at interval 0-100bp (preferred, be 0-50bp; Preferred, be 0-20bp).
In another preference, described goal gene includes, but is not limited to: next to axis protocalcium Fibronectin gene (Paraxial Protocadherin (PAPC)), luciferase gene, fluorescence protein gene.
In another preference, described next to axis protocalcium Fibronectin gene (Paraxial Protocadherin (PAPC)) is the paraxial protocalcium Fibronectin of African Xenopus laevis gene (Xenopus Paraxial Protocadherin (XPAPC)).
In a third aspect of the present invention, a kind of cell (preferred genetically engineered cell) is provided, described cell contains described carrier.
In a fourth aspect of the present invention, the purposes of described nucleotide sequence is provided, described nucleotide sequence is used for as promoter element, instructs the expression of goal gene.
In another preference, described nucleic acid is used to instruct goal gene to express according to phraseology in the next to axis protocalcium Fibronectin genosome.
In another preference, described goal gene includes, but is not limited to: next to axis protocalcium Fibronectin gene (Paraxial Protocadherin (PAPC)), luciferase gene, fluorescence protein gene.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown the molecular cloning and the transcriptional activity analysis of XPAPC upstream dna sequence dna.
(A) uciferase activity of different XPAPC promoter deletion constructs.
(B) detect the XPAPC genome sequence and whether have intron.
Fig. 2 has shown the expression that is expressed in recurrence endogenous XPAPC on the room and time specificity of the GFP mRNA of promoters driven in the transgenic embryos.
(A-E) endogenous XPAPC mRNA is in the expression of each phase (St).
(F-J) the pFL GFP expression in the phase transgenic embryos at the same time.
Wherein, (A-B and F-G) back side up, from vegetative pole;
Wherein, face up dorsal view before (C and H) neurula;
Wherein, (D and I) neurula front is in the left side, and the side is seen;
Wherein, the side of (E and J) tadpole is seen, and the front is in the left side.
Embodiment
The inventor is by extensive and deep research, from the Xenopus laevis genome, be separated to a kind of nucleic acid first, this nucleic acid is positioned at the upstream of the paraxial protocalcium Fibronectin of Africa xenopus gene (XPAPC gene), and it as promoter element, can be instructed the expression of goal gene (as reporter gene).Described nucleic acid comprised instruct XPAPC from primitive gut in early days to the required controlling element of tail bud phase specifically expressing, thereby can be used for research during fetal development the expression regulation situation of specific gene (as XPAPC) and other factors to the expression of specific gene.Finished the present invention on this basis.
As used herein, described " promotor " or " promoter region (territory) " is meant a kind of nucleotide sequence, and the upstream (5 ' end) that it is present in the goal gene encoding sequence usually can be transcribed into mRNA by the guiding nucleus acid sequence.Usually, promotor or promoter region provide RNA polymerase and correct initial recognition site of transcribing necessary other factor.In this article, described promotor or promoter region comprise the variant of promotor, and it is by inserting or deletion regulation and control zone, carry out at random or rite-directed mutagenesis waits and obtains.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, described " being operably connected " is meant functional spatial disposition of two or more nucleic acid region or nucleotide sequence.For example: promoter region is placed in the specific position with respect to the goal gene nucleotide sequence, makes transcribing of nucleotide sequence be subjected to the guiding of this promoter region, thereby promoter region is " operably connected " on this nucleotide sequence.
The genetic expression of promotor and guidance thereof
The inventor is cloned into one section nucleic acid (promotor) that can instruct exogenous gene expression well first from the upstream of XPAPC gene in research process, utilize this promotor to instruct the expression of reporter gene, can simulate the endogenous expression of XPAPC well.In an embodiment of the present invention, the inventor has proved that the expression that utilizes the GFP that described promotor instructs can simulate the expression of endogenous XPAPC fully, illustrate this fragment comprised instruct XPAPC from primitive gut in early days to all required controlling elements of tail bud phase specifically expressing, thereby changed in the present prior art present situation that in the concrete body XPAPC gene is had nucleotide sequence the unknown of regulating and controlling effect.
Therefore, the invention provides a kind of isolating nucleic acid (promotor), described nucleic acid has: the sequence shown in the SEQ ID NO:1, or having the nucleotide sequence shown in the position, (50 ± 49)-(4724 ± 49) among the SEQ ID NO:1, described nucleic acid can be used as the promoter element that instructs destination gene expression.
In addition, the present invention also comprise above-mentioned nucleic acid some have the varient of identical function.Comprise: the sequence shown in (50 ± 49)-(4724 ± 49) positions has more than 95% (preferred 99%) homology and has the nucleic acid that instructs the destination gene expression function among sequence and SEQ ID NO:1 or the SEQ ID NO:1.
Promotor of the present invention can be operatively connected on the goal gene, and this goal gene can be external source (allos) for promotor.Described goal gene can be any nucleotide sequence (as a kind of structural nucleotide sequence) usually; Described goal gene optimized encoding has the albumen of specific function, and for example some has the albumen of key property or function; Perhaps described goal gene is the gene that can bring into play indicative function after transcribing or expressing, as reporter gene.
For example, described goal gene includes but not limited to: Africa xenopus next to axis protocalcium Fibronectin gene, luciferase gene, green fluorescence protein gene.
Promotor of the present invention can also be operably connected on the target gene sequences that is modified, and this goal gene is external source (allos) with respect to promotor.Described goal gene can be modified and produce various desired characteristics.For example, goal gene can be modified increases contents of essential amino acids, improves the translation of aminoacid sequence, change the modification (as phosphorylation site) after translating, outside the translation product transporte to cells, improve proteic stability, insert or delete cell signal etc.
In addition, promotor and goal gene can be designed to reduce specific gene.This generally is to realize that by promotor is connected on the target gene sequences this sequence oppositely is directed with antisense.Those of ordinary skill in the art is familiar with this antisense technology.Any nucleotide sequence can be conditioned by this way.
Promotor of the present invention also can be used for instructing expression of exogenous gene, function of important molecule, the foundation of developmental defect animal model and the gene therapy of developmental defect etc. in growth of research mesoderm and the inner ear growth course.
Promotor of the present invention and target gene sequences can be comprised in the recombinant vectors.
Described recombinant vectors generally comprises (from 5 ' to 3 ' direction): the promotor that the guiding goal gene is transcribed, and goal gene.If desired, described recombinant vectors can also comprise 3 ' transcription terminator, 3 ' polymerized nucleoside acidifying signal, other untranslated nucleotide sequence, transhipment and target nucleotide sequence, resistance selective marker, enhanser or operation.
The method that is used to prepare recombinant vectors is well-known to those skilled in the art.Term " expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, mammalian cell virus or other carriers.In a word, as long as it can duplicate in host and stablize, any plasmid and carrier all are can be adopted.Preferably, described expression vector is a carrier for expression of eukaryon.
Method well-known to those having ordinary skill in the art can be used to make up the expression vector that contains promotor of the present invention and/or target gene sequences.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, described marker gene such as Tetrahydrofolate dehydrogenase, neomycin resistance, hygromycin resistance etc.
Except containing promotor of the present invention, also can contain one or more other promotors in the recombinant vectors.Described other promotor for example is: tissue-specific, composing type or induction type.
As a kind of example of the present invention, described carrier is pGL3-Basic, itself contains the sequence of the plain enzyme gene of coding fluorescence.By transforming, promptly utilize the multiple clone site on the pGL3-Basic, promoter region of the present invention can be building up to the front of luciferase encoding gene, transformed host cell, promotor will activate the expression of luciferase encoding gene, described startup is subjected to the regulation and control of each cis-acting elements of promoter region, has simulated the situation that gene is activated in vivo and transcribes.
Comprise the above-mentioned suitable promotor and the carrier of goal gene, can be used to transform appropriate host cell, or be transformed in zygote or the embryo, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria, yeast, the histocyte of animal etc.Zygote or embryo can be the zygote or the embryos of amphibian animal, for example are the zygote or the embryos of African Xenopus laevis.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser or host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The method that recombinant DNA transforms zygote or embryo also is known in the art, can adopt microinjection technique usually.For example can carry out transgeneic procedure according to described methods of (1996) .Transgenic Xenopus embryos fromsperm nuclear transplantations reveal FGF signaling requirements duringgastrulation.Development 122:3173-3183 such as Kroll KL.Can carry out linearization in advance as genetically modified plasmid.
In example of the present invention, under the guidance of described promotor, luciferase (Luciferase) gene and green fluorescent protein (GFP) gene are expressed well.Therefore as seen, promotor of the present invention has important use value in the research of gene expression regulation.
In example of the present invention, after the encoding sequence replacement of encoding sequence with luciferase in the XPAPC promoter luciferase analysis plasmid of total length, obtained being used to prepare transgenosis Xenopus laevis embryo's reporter plasmid with green fluorescent protein.The inventor has prepared the transgenosis Xenopus laevis embryo of different times and has carried out hybridization in situ experiment with the probe that reacts the GFP expression.The result shows, transgenosis Xenopus laevis embryo at different times, the expression pattern of the guidable GFP of XPAPC promotor and endogenous XPAPC expression pattern are closely similar, have comprised in the XPAPC promotor that this explanation inventor obtains and have known that XPAPC is at needed all the important cis-acting elements of Xenopus laevis embryo early expression.
Major advantage of the present invention is:
Disclose a kind of promotor that instructs destination gene expression, utilize promotor of the present invention can study the expression of the expression regulation situation of specific gene during fetal development (as XPAPC) and other factors (as some signal paths) specific gene.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1 embryo culture and transgenic method
Africa xenopus embryo culture operation: (2004) such as the cultivation of zygote such as Fang PF, described in Multiplesignaling pathways control Tbx6 expression during Xenopus myogenesis.Acta Biochim Biophys Sin (Shanghai) 36:390-396, by stages according to Nieuwkoop and Faber (Normal Table of Xenopus Laevis, Garland Publishing, 1994) described in.
Africa xenopus transgenic embryos preparation: carry out transgeneic procedure according to described methods of (1996) .Transgenic Xenopusembryos from sperm nuclear transplantations reveal FGF signalingrequirements during gastrulation.Development 122:3173-3183 such as Kroll KL, obtained genetically modified embryo.Digest with linearization with SmaI as genetically modified plasmid.Each plasmid has all carried out 3 and has taken turns independently transgeneic procedure.
The clone of embodiment 2XPAPC 5 ' upstream regulatory sequence
Because the genome sequence of Africa xenopus is not also finished by order-checking, the upstream regulatory sequence that the inventor has cloned XPAPC by unique design is specific as follows:
Genomic dna separates: get 2g bull Xenopus laevis fresh liver tissue, it is abundant to put into mortar adding liquid nitrogen grinding, with phenol/chloroform method extracting twice, gets the supernatant ethanol sedimentation, and 75% ethanol is washed one time, and drying is dissolved in the deionized water, and spectrophotometer is quantitative.
Connect: get 1 μ g genomic dna and spend the night with the EcoR I of 10U digestion, cancellationization back 1 μ g genomic dna is connected for 16 ℃ with the T4DNA ligase enzyme with 50mM PCR joint and spends the night, and afterwards with the ethanol sedimentation recovery, is dissolved in the 20 μ l deionized waters.
The primer that PCR adopts is as follows:
AP1(SEQ?ID?NO：2)：5’-GTAATACGACTCACTATAGGC-3’；
AP2(SEQ?ID?NO：3)：5’-ACAATAGGGCACGCGTGGT-3’；
GSP1(SEQ?ID?NO：4)：5’-CAACACTGCAATTACAGTGCCAGGGGGTTCTTCTTC-3’；
GSP2(SEQ?ID?NO：5)：5’-GAGAAGCAGCATCTTGAAGAATCAAAGTTGCACC-3’。
The primer of AP1 and AP2 is based on genome walking (Genome Walker) adapter system.
Two-wheeled joint PCR (Adaptor PCR) separates amplification XPAPC 5 ' upstream sequence:
The first round, adopt 50 μ l systems, consumption is as follows:
10 * damping fluid, 5 μ l;
dNTP 250μl；
GSP1 10pM；
AP1 10pM；
Connected genomic dna 100ng after the digestion of joint;
LA Taq polysaccharase 2.5U;
Deionized water complements to 50 μ l.
The PCR program:
94 ℃ of sex change 3 minutes;
10 circulations of the first step: 94 ℃ of sex change 30 seconds;
Anneal 70 ℃ 30 seconds;
Extend 72 ℃ 5 minutes;
Second step 30 circulations: 94 ℃ of sex change 30 seconds;
Anneal 60 ℃ 1 minute;
Extend 72 ℃ 5 minutes;
Extend at last 72 ℃ 10 minutes.
Second takes turns PCR: first round PCR product is diluted 100 times in following ratio application of sample, and consumption is as follows:
10 * damping fluid (adds Mg
2+) 5 μ l;
dNTP 250μl；
GSP2 10pM；
AP2 10pM；
100 times of 100ng of first round PCR product dilution;
LA Taq polysaccharase 2.5U;
Deionized water complements to 50 μ l.
Carry out PCR with first round same program.
Second product of taking turns PCR is separated with 1% agarose gel, identify and find that there is single band at the 4.3kb place, rubber tapping is reclaimed, the T-easy carrier (Promega) of packing into, order-checking.
As a result, length is that the dna fragmentation of 4914bp is cloned, shown in SEQ ID NO:4.Its 5 ' zone (referring to (1998) such as Kim SH, The role ofparaxial protocadherin in selective adhesion and cell movements of themesoderm during Xenopus gastrulation.Development 125:4681-4690) with disclosed Xenopus PAPC gene had 143bp's is overlapping.
Embodiment 3 contains the structure of the carrier of promotor and reporter gene
Behind the pcr amplified fragment ordinary method purifying of aforementioned acquisition, obtain the fragment that the XhoI/HindIII restriction enzyme site is carried at 1-4773 bit sequence among the SEQ IDNO:1 and two ends by conventional PCR method, be inserted into the PGL3-Basic (Promega of XhoI/HindIII digestion, carry the luciferase encoding gene) in, the p-4773Luc construct obtained.
The inventor also utilizes the XbaI/HindIII enzyme to cut digestion p-4773Luc, and luciferase gene is wherein removed, and reclaims the plasmid through processing; Encoding sequence (the GFP encoding sequence is to downcut with XbaI and HindIII from pCS2-GFP plasmid (available from Ralph Rupp laboratory)) with green fluorescent protein (GFP) is encased in through aforementioned through the carrier of processing simultaneously, obtains the used plasmid p-4773GFP of transgenosis.
The inventor has also made up the fragment that is made of 1-2920 bit sequence among the SEQ ID NO:1, adopt aforementioned same procedure that this sequence is respectively charged in PGL3-Basic and green fluorescent protein (GFP) reporter plasmid, thereby obtain test plasmid p-2920Luc and p-2920GFP.
The luciferase reporting plasmid of above-mentioned structure will be used to determine the activity of reporter gene, and the green fluorescent protein reporter plasmid will be used to carry out the experiment of Xenopus laevis transgenic embryos.
The transcriptional control analysis of embodiment 4 promotors
For carrying out the luciferase analysis, the 2nl solution (solvent is a DEPC water) that will contain mark reporter gene pRL-SV40 (available from Promega) in different luciferase test plasmids of 25pg and the 25pg is injected into the animal pole of four cell stage.
Whether has transcriptional activity in order to analyze resulting XPAPC 5 ' upstream sequence, p-4773Luc, p-2920Luc carrier, PGL3-Basic empty carrier (each 25pg with aforementioned structure, 2nl) with confidential reference items contrast pRL-SV40 (25pg, 2nl) be expelled to respectively in the Xenopus laevis embryo animal pole of four cell stage, separating animal's polar cap about the 8.5th phase, be cultured to for 12.5 phases then, the uciferase activity of examining report gene.The luciferase analysis is carried out with Dual-Luciferase Reporter Assay System (Promega).
The results are shown in Figure 1A, show that XPAPC 5 ' upstream sequence fragment has transcriptional activity.
In eukaryote, intron (intron), particularly first intron also might participate in the regulation and control of genetic expression.In order whether there to be intron in the genome sequence that detects XPAPC, the inventor has carried out genome PCR.
The result is adding the primer (upstream: TTG TTT TAA ATG ACT GCAGGT CTG GAA GGA (SEQ ID NO:6) of specific combination in XPAPC open reading frame (open readingframe) zero position and final position shown in Figure 1B; The downstream: in CAG TTC CCA TTT TCT GGACCC TTG TTG A (SEQ ID NO:7)) the PCR reaction, the fragment that a length is only arranged is 3.6kb can be amplified.Show XPAPC genome sequence and other the same intron that do not comprise of some former cadherins by this fragment being carried out dna sequencing.
Whether can instruct the expression of reporter gene GFP in the Africa xenopus embryo in order to detect resulting XPAPC 5 ' upstream regulatory sequence, the inventor utilizes SmaI will comprise plasmid p-4773GFP (pFL-GFP) linearizing of upstream regulatory sequence, be transferred among the Xenopus laevis embryo, then by the expression of in situ hybridization examining report gene GFP in embryo development procedure, and detect the expression of unconverted embryo's endogenous XPAPC.The method with whole in situ hybridization of transcribing of XPAPC and GFP is respectively with XPAPC or GFP probe in detecting.
The preparation of mRNA template is according to (2004) such as document Fang PF, described in Multiple signalingpathways control Tbx6 expression during Xenopus myogenesis.ActaBiochim Biophys Sin (Shanghai) 36:390-396.Embryo's in situ hybridization utilizes the XPAPC of digoxin (DIG)-mark or GFP probe, and (the XPAPC probe is with reference to Kim SH etc., described in the The roleof paraxial protocadherin in selective adhesion and cell movements ofthe mesoderm during Xenopus gastrulation.Development 125:4681-4690.; GFP probe system is with after the pCS2-GFP linearizing, obtain with the t7 rna polymerase in-vitro transcription), according to the carrying out of being put down in writing among the Harland RM.1991.In situ hybridization:an improvedwhole-mount method for Xenopus embryos.Methods Cell Biol 36:685-695.The hybridization embryo handles down with sodium hypochlorite solution (0.5 * SSC and 5% methane amide that contain 1% hydrogen peroxide) luminescent lamp.
Found that in the Africa xenopus embryo, XPAPC begins previous halfhour blastaea from former bowel movement the earliest and begins to express (9.5 phases (St9.5)) late period.In this period, using integral embryo in situ hybridization can detect the expression (Fig. 2 A) of XPAPC at back of the body method, edge band.In early stage (10 phase) of gastrula, the expression of XPAPC expands to whole peripheral zone and presents the many and few pattern (Fig. 2 B) in abdomen side in back of the body side.Along with the carrying out of former bowel movement, (notochord notochord) germinates and reduces XPAPC along with axial mesoderm in the expression of back of the body side's center line.When the mesoderm cortex 13 phases by after the complete curls inward, XPAPC mRNA expression forwardly have one clearly the border with head and trunk mesoderm separately (Fig. 2 C).In case body segment forms (somitogenesis) beginning, the expression of XPAPC will be reduced in sophisticated body segment and banded expression meeting is moved (Fig. 2 D) to the embryo rear, such circulation form to finish just to stop up to body segment, and the expression of XPAPC after this maintains (24 phase) (Fig. 2 E) on the tip of afterbody.Except mesoblastic expression, since 17 phases, XPAPC also is about to form the position expression (Fig. 2 D) of cochlea in the head both sides, thereafter along with the growth and the curls inward of cochlea, the expression of XPAPC also is confined to the zone of cochlea more, after ear cup formed, XPAPC can be expressed in the inside (Fig. 2 E) of cup-like structure.
Having carried out transgeneic procedure linearizing pFL-GFP reporter plasmid is incorporated among the Xenopus laevis embryo in the genome, the expression pattern of GFP no matter be in former bowel movement mesoderm or among the cochlea of growing, all express closely similar (Fig. 2 F is to J) with endogenous XPAPC.Illustrate the promotor with 1-4773 bit sequence among the SEQ ID NO:1 comprised instruct XPAPC from primitive gut in early days to the required controlling element of tail bud phase specifically expressing.
In addition, the inventor utilizes the p-2920GFP plasmid by similar method, and checking finds that the promotor of 1-2920 bit sequence among the SEQ IDNO:1 can instruct GFP to express, but expression is different with endogenous XPAPC expression, and this causes owing to lacking some controlling element in this sequence.
In addition, the inventor has verified that by similar method the nucleic acid that is made of 3-4769 bit sequence among the SEQ ID NO:1 also has the activity that is similar to the 1-4773 bit sequence.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉Africa xenopus XPAPC gene promotor and tissue-specific enhancer thereof
<130>080302
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<170>PatentIn?version?3.3
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＜213〉Africa xenopus (Xenopus laevis)
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ctaaatagac?tggccttttg?ggtgttttct?ggacattatc?tctaagaagg?accatttaat 960
taaatgacgg?aaggatttgc?accaaaagac?ttggacaatg?catacaccat?atggaaaaat 1020
gtaagctcac?atggagcaaa?tgttgtacaa?cttactctcg?atatttgtct?gatattaaat 1080
ctgggtcaaa?tgttcgtaaa?aaatcatctc?tctctacaac?aaattctgca?gaatacattg 1140
ggcttgctag?ttctcctacc?atgggcttgt?tgtacattta?ggaaaggctt?tggggtattt 1200
aataccattt?gttcaacaag?taggccttgt?atcctagttg?aaatatagac?attgtggcta 1260
ataagacctg?aggcactagg?gatataatta?cctattcagt?tctaattgat?gatcttaaga 1320
agggtactct?aatcaatgca?gtgttttgtg?tactcaagtg?aatagccttg?taatggacga 1380
ggacctcctc?ctgccttgaa?tgggaaattg?taagtcttat?tcttttagta?gttgtttatt 1440
aggattgtct?acacacactt?ccacccactc?attctgtaca?aatatacaca?tataagcaca 1500
agttatattt?attgccatgc?ctaaatgctt?gtctgtttct?atattacaga?ctgtgtattg 1560
gctataatta?aagggaatgt?tgattttttt?tatcagggtt?ttttttcaga?gcggcactgt 1620
ttatgcattg?tgtttgttgt?gcaatccaca?gatgtcaggc?actgggctat?actagtcgtt 1680
ggagacaaaa?ggtagacagc?aggcaaatac?aacatttaag?gcaatgccat?agggacaatt 1740
atgcttgaac?tgcattagtg?attatcactg?acctgcaacg?ctttccatcc?aagttgatgg 1800
gatgcaactt?ctagtacttg?tgtgccatgc?ataggggtaa?aacaacagtg?gaagcagacc 1860
ctgtgactgc?tgaagagccc?ccagagtttt?aggggtctag?tggagcagta?attaataagc 1920
gctttcaata?tatacttttg?aaaattgtct?tattctgagg?ttcagtgaac?cataaaatga 1980
ttttcatgac?tcaataactt?ctagtccagt?ggccatgcac?tgttttgctg?catatggagc 2040
atggactaat?tttgcgctgt?atagctgttc?ttctattcca?tttccaatta?aagtattgac 2100
ctgcacgtgg?catgtgtatt?tggtaatact?ggtatgggaa?cccttaccca?gaacctgtta 2160
tccagatagt?tccgatttac?aggaaggcca?tgttccatag?actccatttt?atctaaataa 2220
tccagatttt?tatcaactat?ttcctttatc?tctgtaaggt?tacagacaca?cgctaagatt 2280
cagggagatt?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?ttaccctaat?aataaaatag 2580
tagcttgtac?ttgatccaaa?ctaagatata?attaatcctt?attgaaggca?aatccaagct 2640
attgggttta?ttaaatgttt?acatgatttt?ctagtatact?taaggtatga?agatccaaat 2700
tacggaaaaa?tcagttaccc?agaaaacccc?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?acatatttat?atttttatta?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?ggtttattta?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?atttttagtt?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?gccacactgt?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
acaatagggc?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 (10)
1. an isolated nucleic acid sequences is characterized in that, described nucleotide sequence is selected from down group:
(1) has the nucleotide sequence shown in the SEQ ID NO:1;
(2) has the nucleotide sequence of sequence shown in the position, (50 ± 49)-(4724 ± 49) among the SEQ ID NO:1; Or
(3) nucleotide sequence with (1) or (2) qualification has 95% above homogeny and has the nucleotide sequence that instructs the destination gene expression function.
2. nucleotide sequence as claimed in claim 1 is characterized in that, in (2) item, has the nucleotide sequence of sequence shown in the position, (20 ± 19)-(4754 ± 19) among the SEQ ID NO:1.
3. a carrier is characterized in that, described carrier contains the described nucleotide sequence of claim 1, as promoter element.
4. carrier as claimed in claim 3 is characterized in that described carrier also contains the target gene sequences that is operably connected with described nucleotide sequence.
5. carrier as claimed in claim 4 is characterized in that, described nucleotide sequence and target gene sequences be 0-100bp at interval.
6. carrier as claimed in claim 4 is characterized in that, described goal gene comprises: next to axis protocalcium Fibronectin gene, luciferase gene or fluorescence protein gene.
7. a cell is characterized in that, described cell contains the described carrier of claim 3.
8. the purposes of the described nucleotide sequence of claim 1 is characterized in that, described nucleotide sequence is used for as promoter element, instructs the expression of goal gene.
9. purposes as claimed in claim 8 is characterized in that, described nucleotide sequence is used to instruct goal gene to express according to phraseology in the next to axis protocalcium Fibronectin genosome.
10. purposes as claimed in claim 8 is characterized in that, described goal gene comprises: next to axis protocalcium Fibronectin gene, luciferase gene or fluorescence protein gene.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100330862A CN101492673B (en) | 2008-01-25 | 2008-01-25 | Africa xenopus XPAPC gene promotor and tissue specificity enhancer |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100330862A CN101492673B (en) | 2008-01-25 | 2008-01-25 | Africa xenopus XPAPC gene promotor and tissue specificity enhancer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101492673A true CN101492673A (en) | 2009-07-29 |
| CN101492673B CN101492673B (en) | 2012-01-11 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2008100330862A Expired - Fee Related CN101492673B (en) | 2008-01-25 | 2008-01-25 | Africa xenopus XPAPC gene promotor and tissue specificity enhancer |
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| CN (1) | CN101492673B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002500A (en) * | 2010-11-02 | 2011-04-06 | 浙江大学 | Xenopus leavis dmrt5 gene nervous specificity regulating and controlling element and construction method for carrier thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5798224A (en) * | 1992-12-29 | 1998-08-25 | Doheny Eye Institute | Nucleic acids encoding protocadherin |
| WO2001098483A1 (en) * | 2000-06-22 | 2001-12-27 | Zoegene Corporation | Gene encoding novel protocadherin-like protein |
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2008
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002500A (en) * | 2010-11-02 | 2011-04-06 | 浙江大学 | Xenopus leavis dmrt5 gene nervous specificity regulating and controlling element and construction method for carrier thereof |
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| Publication number | Publication date |
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| CN101492673B (en) | 2012-01-11 |
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