CN105779457B - Cardiac muscle cell's specific promoter and its application - Google Patents
Cardiac muscle cell's specific promoter and its application Download PDFInfo
- Publication number
- CN105779457B CN105779457B CN201610374248.3A CN201610374248A CN105779457B CN 105779457 B CN105779457 B CN 105779457B CN 201610374248 A CN201610374248 A CN 201610374248A CN 105779457 B CN105779457 B CN 105779457B
- Authority
- CN
- China
- Prior art keywords
- sequence
- cmrp
- nucleotide
- pgl3
- dna molecular
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
- C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
Abstract
The invention discloses cardiac muscle cell's specific promoter and its applications.Cardiac muscle cell's specific promoter disclosed by the invention, it is following DNA fragmentations a) or b) or c): a) 927-1501 nucleotide sequences of the 3 ' ends at least containing sequence 1 in ordered list, and extends since the 927th of sequence 1, according to the nucleotide sequence of sequence 1 to 5 ' ends of sequence 1, obtain any one DNA fragmentation that length is 575 to 1501bp；The DNA molecular has promoter function；B) nucleotide sequence and a) limited has 75% or 75% or more identity, and the DNA fragmentation with promoter function；C) under strict conditions with the nucleotide sequence hybridization that a) or b) limits, and the DNA fragmentation with promoter function.It is demonstrated experimentally that promoter of the invention has cardiac muscular tissue's specificity and heartspecific.
Description
Technical field
The present invention relates to field of biotechnology cardiac myocyte specific promoter and its applications.
Background technique
Since the 21th century, cardiovascular disease has become the important diseases for threatening human health.In the world
Interior, raised trend year by year is presented in the disease incidence of cardiovascular disease, in China, cardiovascular patient about 2.3 hundred million people, every year
Die of about 3,000,000 people of cardiovascular disease, it may be said that cardiovascular disease has become the first killer for threatening human health.Mental and physical efforts
Failure is one of the main reason for cardiovascular disease leads to death, and there are about 40% cardiovascular diseases to develop into heart failure,
Therefore, the reason of verifying heart failure morbidity, mechanism, and find appropriate treatment heart failure target site meaning it is very great.
The pathogenesis of heart failure is extremely complex, has had many researchers to be set out from different angles to illustrate the heart
The development process of force failure.Research has shown that have a large amount of intracellular molecules signal path to take part in this process, such as: pressure
Load is a key factor for causing heart failure, and pathologic pressure passes through G-protein coupling receptor, calmodulin-dependent
Protein kinase (CAMKII), mitogen-activated protein kinase (MAPKs), protein phosphatase calcineurin and transcription factor
Cardiac muscle cell's enhancement factor 2 (MEF2), active t cell nuclear factor (NFAT) this signal path, finally causes heart failure dependency basis
The variation of the expression of cause, so as to cause the hypertrophy of cardiac muscle cell, failure.It is phase that these complicated signal paths, which have quite a few,
Mutually overlapping with it is interactional, most probably become heart failure in the overlapped node of unlike signal access, such as GSK3 β, HDACs
The important target spot for the treatment of.
When carrying out gene therapy to heart disease, how therein important one is become to heart targeting to completion
Ring is badly in need of a kind of promoter with heart or cardiac myocytespecific at present.
Summary of the invention
Technical problem to be solved by the invention is to provide with cardiac myocytespecific promoter function DNA molecular.
In order to solve the above technical problems, present invention firstly provides the DNA for deriving from rat (Rattus norvegicus)
Molecule, the entitled CMRP of the DNA molecular, CMRP have a cardiac myocytespecific promoter function, CMRP be it is following a) or b)
Or DNA fragmentation c):
A) 3 ' 927-1501 nucleotide sequences of the end at least containing sequence 1 in ordered list, and from the of sequence 1
927 start, according to sequence 1 nucleotide sequence to sequence 15 ' end extend, obtain length be 575 to 1501bp it is any
One DNA fragmentation；The DNA fragmentation has promoter function；
B) nucleotide sequence and a) limited has 75% or 75% or more identity, and the DNA with promoter function
Segment；
C) under strict conditions with the nucleotide sequence hybridization that a) or b) limits, and the DNA fragmentation with promoter function.
Wherein, sequence 1 is made of 1501 nucleotide.
In above-mentioned DNA molecular, the stringent condition is hybridized simultaneously at 68 DEG C in 2 × SSC, the solution of 0.1%SDS
It washes film 2 times.In the solution of each 5min 0.5 × SSC, 0.1%SDS, hybridizes at 68 DEG C and wash film 2 times.Each 15min.
Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90%, 95% or more identity.
Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation
Method is mutated promoter nucleotide sequence of the invention.Those are by manually modified, with isolated with the present invention
Promoter nucleotide sequence 70% or higher identity nucleotide, as long as the promoter for maintaining expression target gene is living
Property, it is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair
Bright promoter nucleotide sequence has 75% or higher or 85% or higher or 90% or higher or 95% or higher same
The nucleotide sequence of property.Identity can with the naked eye or computer software is evaluated.It is two or more using computer software
Identity between sequence can be indicated with percentage (%), can be used to evaluate the identity between correlated series.
In above-mentioned DNA molecular, last nucleotide of CMRP is the 1501st of sequence 1.
In above-mentioned DNA molecular, CMRP can also be following A 1)-A8) in any DNA fragmentation:
A1) 913-1501 nucleotide sequences of the 3 ' ends of nucleotide sequence at least containing sequence 1 in ordered list, and
Since the 913rd of sequence 1, according to sequence 1 nucleotide sequence to sequence 15 ' end extend, obtain length be 589 to
Any one DNA fragmentation of 1501bp；
A2) 888-1501 nucleotide sequences of the 3 ' ends of nucleotide sequence at least containing sequence 1 in ordered list, and
Since the 888th of sequence 1, according to sequence 1 nucleotide sequence to sequence 15 ' end extend, obtain length be 614 to
Any one DNA fragmentation of 1501bp；
A3) 870-1501 nucleotide sequences of the 3 ' ends of nucleotide sequence at least containing sequence 1 in ordered list, and
Since the 870th of sequence 1, according to sequence 1 nucleotide sequence to sequence 15 ' end extend, obtain length be 632 to
Any one DNA fragmentation of 1501bp；
A4) 848-1501 nucleotide sequences of the 3 ' ends of nucleotide sequence at least containing sequence 1 in ordered list, and
Since the 848th of sequence 1, according to sequence 1 nucleotide sequence to sequence 15 ' end extend, obtain length be 654 to
Any one DNA fragmentation of 1501bp；
A5) 827-1501 nucleotide sequences of the 3 ' ends of nucleotide sequence at least containing sequence 1 in ordered list, and
Since the 827th of sequence 1, according to sequence 1 nucleotide sequence to sequence 15 ' end extend, obtain length be 675 to
Any one DNA fragmentation of 1501bp；
A6) 789-1501 nucleotide sequences of the 3 ' ends of nucleotide sequence at least containing sequence 1 in ordered list, and
Since the 789th of sequence 1, according to sequence 1 nucleotide sequence to sequence 15 ' end extend, obtain length be 713 to
Any one DNA fragmentation of 1501bp；
A7) 819-1501 nucleotide sequences of the 3 ' ends of nucleotide sequence at least containing sequence 1 in ordered list, and
Since the 819th of sequence 1, according to sequence 1 nucleotide sequence to sequence 15 ' end extend, obtain length be 683 to
Any one DNA fragmentation of 1501bp；
A8) 1051-1501 nucleotide sequences of the 3 ' ends of nucleotide sequence at least containing sequence 1 in ordered list, and
And extend since the 1051st of sequence 1, according to the nucleotide sequence of sequence 1 to 5 ' ends of sequence 1, obtaining length is 451
To any one DNA fragmentation of 1501bp.
In above-mentioned DNA molecular, CMRP can be following 1) -10) in any one:
1) DNA molecular shown in 927-1051 nucleotide of sequence 1 in sequence table；
2) DNA molecular shown in 913-1051 nucleotide of sequence 1 in sequence table；
3) DNA molecular shown in 888-1051 nucleotide of sequence 1 in sequence table；
4) DNA molecular shown in 870-1051 nucleotide of sequence 1 in sequence table；
5) DNA molecular shown in 848-1051 nucleotide of sequence 1 in sequence table；
6) DNA molecular shown in 827-1051 nucleotide of sequence 1 in sequence table；
7) DNA molecular shown in 713-1051 nucleotide of sequence 1 in sequence table；
8) DNA molecular shown in 683-1051 nucleotide of sequence 1 in sequence table；
9) DNA molecular shown in 451-1051 nucleotide of sequence 1 in sequence table；
10) DNA molecular shown in 1-1051 nucleotide of sequence 1 in sequence table.
In order to solve the above technical problems, the present invention also provides the biomaterial containing CMRP, under the biomaterial is
State B1) any one of to B13):
B1) containing the expression cassette of CMRP；
B2) containing the recombinant vector of CMRP；
B3) contain B1) recombinant vector of the expression cassette；
B4) containing the recombinant microorganism of CMRP；
B5) contain B1) recombinant microorganism of the expression cassette；
B6) contain B2) recombinant microorganism of the recombinant vector；
B7) contain B3) recombinant microorganism of the recombinant vector；
B8) the transgenetic animal cell system containing CMRP；
B9) contain B1) the transgenetic animal cell system of the expression cassette；
B10) the transgenic animals tissue containing CMRP；
B11) contain B1) the transgenic animals tissue of the expression cassette；
B12) containing the transgenic animal organ of CMRP；
B13) contain B1) transgenic animal organ of the expression cassette.
In above-mentioned biomaterial, the transgenetic animal cell system, transgenic animals tissue and the transgenosis are dynamic
Sundries official does not include the propagation material of animal.
In above-mentioned biomaterial, the expression cassette can be started the target gene and tanscription termination of expression by CMRP, CMRP
Sequence composition；CMRP is connect with functional way with the target gene, and the target gene and the transcription terminator
Connection.In one embodiment of the invention, the target gene is specially luciferase gene (luc).
In the recombinant vector, by the expression of CMRP starting target gene.In one embodiment of the invention, described heavy
Group carrier is specially the recombinant vector obtained in the multiple cloning sites insertion CMRP of pGL3-Basic carrier.The multiple cloning sites
Specially restriction enzyme enzyme recognition site MluI and NheI.The target gene is specially luciferase gene (luc).
Above-mentioned expression cassette or recombinant vector can pass through pronuclear microinjection method, embryonic stem cell mediated method, reverse transcription disease
Poisonous carrier method, the gene transfer of Sperm-mediated, nuclear transfer transgemic approach, body-cell neucleus transplanting method, mitochondria mediated method etc. are conventional
Biological method transformed animal organ or tissue or cell obtain transgenetic animal cell or tissue or organ.
In order to solve the above technical problems, the present invention also provides any applications in following M1-M4:
M1, CMRP are as the application in promoter；
M2, CMRP are as the application in specific heart promoter；
M3, CMRP are as the application in myocardium specific promoter；
M4, CMRP are as the application in cardiac muscle cell's specific promoter.
In order to solve the above technical problems, the present invention also provides any applications in following N1-N3:
N1, CMRP start the application in destination gene expression in heart；
N2, CMRP start the application in destination gene expression in cardiac muscle；
N3, CMRP start the application in destination gene expression in cardiac muscle cell.
Start answering in destination gene expression in animal in order to solve the above technical problems, the present invention also provides CMRP
With.
In above-mentioned application, the expression can be expressed for specific heart, such as cardiac muscle cell's specifically expressing.At of the invention one
In specific embodiment, the cardiac muscle cell concretely neonatal rat myocardial cell.
In order to solve the above technical problems, the present invention also provides CMRP to cultivate transgenic animals (such as anti-heart disease
Transgenic animals) in application.
In the present invention, the animal can be terrestrial animal, such as mammal.
It is demonstrated experimentally that promoter and difference truncated segment --- CMRP-575, CMRP-589, CMRP- of the invention
614, CMRP-632, CMRP-654, CMRP-675, CMRP-789, CMRP-819, CMRP-1051 and CMRP can start report
Gene luc (luciferase) is expressed in cardiac muscle cell, and also confirms that the expression luc of these promoters starting reporter gene is (glimmering
Light element enzyme) there is cardiac myocytespecific, illustrate that promoter of the invention has good cardiac muscular tissue's specificity and heart special
It is anisotropic.Promoter of the invention can be used for cultivating transgenic animals (transgenic animals of such as anti-heart disease), and of the invention opens
Mover has certain application prospect in life science field and heart disease field of gene.
Detailed description of the invention
Fig. 1 is the testing result of the relative luciferase activity of different recombinant cells.Wherein, 1501 C-pGL3- is indicated
CMRP, 533 indicate C-pGL3-CMRP-533,555 indicate C-pGL3-CMRP-555,575 indicate C-pGL3-CMRP-575,589
Indicate C-pGL3-CMRP-589,614 indicate C-pGL3-CMRP-614, and 632 indicate C-pGL3-CMRP-632, and 654 indicate C-
PGL3-CMRP-654,675 indicate C-pGL3-CMRP-675, and 789 indicate C-pGL3-CMRP-789, and 819 indicate C-pGL3-
CMRP-819,1051 indicate that C-pGL3-CMRP-1051, Con indicate C-pGL3-Basic.* indicates relative luciferase activity
It is extremely significant to be lower than C-pGL3-CMRP.
Fig. 2 is the relative luciferase activity of C-pGL3-CMRP, Hek293-pGL3-CMRP and Hela-pGL3-CMRP.
Wherein, NRCM indicates that C-pGL3-CMRP, Hek293 indicate that Hek293-pGL3-CMRP, Hela indicate Hela-pGL3-CMRP.
Specific embodiment
Heretofore described promoter nucleotide sequence can be wherein one or more nucleotide and occur to replace, lack
The nucleotide sequence of mistake, insertion or inversion, i.e., the artificial mutant of separated nucleotide sequence or " natural " mutant, it is protected
Stay its promoter function；It can also be that the nucleotide sequence and other promoter sequences or promoter region guard regulating and controlling sequence
The fusion sequence of (" motif " or " box ").Heretofore described " promoter activity " refers to when expressing with certain gene
Mode is connected to the downstream of promoter, and imported into host, which shows to have and produce in host or outside host
When the ability and function of the gene product, which has starting activity.In general, being that will encode to be easy qualitative or quantitative detection
The gene (for example, reporter gene) of protein be connected to the downstream of the promoter, by channel genes host, and detect institute
The protein of expression, it may be determined that the activity of specific promoter or with the presence or absence of the promoter or the effect of the promoter.This hair
Conversion described in bright refer to it is well known in the prior art, foreign gene can be imported any of zooblast or animal tissue
A kind of Animal transformation methods, such as pronuclear microinjection method, embryonic stem cell mediated method, retroviral vector method, Sperm-mediated
Gene transfer, nuclear transfer transgemic approach, body-cell neucleus transplanting method, mitochondria mediated method etc..
Term " sequence with 75% or 75% or more identity " refers to compared with the nucleotide sequence of above-mentioned promoter
There are 75% or 75% or more the same or similar nucleic acid sequence of nucleotide sequence or nucleotide sequence, these sequences are with basic phase
With mode play a role and the lung tissue of gene downstream can be driven specific expressed, sequence in they and above-mentioned promoter
The difference of column may be due to the modification or mutation in partial structurtes, including artificial mutation and unartificial mutation.
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Carrier pGL3-Basic in following embodiments is promega Products, catalog number E1751.
The identification of embodiment 1, promoter CMRP cardiac myocytespecific promoter function
The present invention provides from SD rat (Rattus norvegicus), (it is limited that tonneau China experimental animal is tieed up in Beijing
Company) DNA molecular, the entitled CMRP of the DNA molecular, CMRP have cardiac myocytespecific promoter function, CMRP's
Sequence is as shown in sequence 1 in sequence table, and the 5 ' ends that wherein the 1st of sequence 1 is CMRP, last holds for the 3 ' of CMRP, sequence
Column 1 are made of 1501 nucleotide.
CMRP is truncated, following DNA molecular: CMRP-533, CMRP-555, CMRP-575, CMRP- is respectively obtained
589,CMRP-614,CMRP-632,CMRP-654,CMRP-675,CMRP-789,CMRP-819,CMRP-1051.Each DNA molecular
Sequence it is as follows:
The sequence of CMRP-533 is the position 969-1051 of sequence 1；
The sequence of CMRP-555 is the position 947-1051 of sequence 1；
The sequence of CMRP-575 is the position 927-1051 of sequence 1；
The sequence of CMRP-589 is the position 913-1051 of sequence 1；
The sequence of CMRP-614 is the position 888-1051 of sequence 1；
The sequence of CMRP-632 is the position 870-1051 of sequence 1；
The sequence of CMRP-654 is the position 848-1051 of sequence 1；
The sequence of CMRP-675 is the position 827-1051 of sequence 1；
The sequence of CMRP-789 is the position 713-1051 of sequence 1；
The sequence of CMRP-819 is the position 683-1051 of sequence 1；
The sequence of CMRP-1051 is the position 451-1051 of sequence 1.
1, the building of recombinant vector
DNA fragmentation between the MluI of carrier pGL3-Basic and NheI identification sequence is replaced with into CMRP shown in sequence 1,
Recombinant vector is obtained, which is named as pGL3-CMRP, in pGL3-CMRP, the identification sequence at the 5 ' ends and MluI of CMRP
Column are connected, and 3 ' ends are connected with the identification sequence of NheI.
DNA fragmentation between the MluI of carrier pGL3-Basic and NheI identification sequence is replaced with into CMRP-533, obtains weight
The recombinant vector is named as pGL3-CMRP-533 by group carrier, and in pGL3-CMRP-533, the 5 ' ends of CMRP-533 are with MluI's
Identify that sequence is connected, 3 ' ends are connected with the identification sequence of NheI.
DNA fragmentation between the MluI of carrier pGL3-Basic and NheI identification sequence is replaced with into CMRP-555, obtains weight
The recombinant vector is named as pGL3-CMRP-555 by group carrier, and in pGL3-CMRP-555, the 5 ' ends of CMRP-555 are with MluI's
Identify that sequence is connected, 3 ' ends are connected with the identification sequence of NheI.
DNA fragmentation between the MluI of carrier pGL3-Basic and NheI identification sequence is replaced with into CMRP-575, obtains weight
The recombinant vector is named as pGL3-CMRP-575 by group carrier, and in pGL3-CMRP-575, the 5 ' ends of CMRP-575 are with MluI's
Identify that sequence is connected, 3 ' ends are connected with the identification sequence of NheI.
DNA fragmentation between the MluI of carrier pGL3-Basic and NheI identification sequence is replaced with into CMRP-589, obtains weight
The recombinant vector is named as pGL3-CMRP-589 by group carrier, and in pGL3-CMRP-589, the 5 ' ends of CMRP-589 are with MluI's
Identify that sequence is connected, 3 ' ends are connected with the identification sequence of NheI.
DNA fragmentation between the MluI of carrier pGL3-Basic and NheI identification sequence is replaced with into CMRP-614, obtains weight
The recombinant vector is named as pGL3-CMRP-614 by group carrier, and in pGL3-CMRP-614, the 5 ' ends of CMRP-614 are with MluI's
Identify that sequence is connected, 3 ' ends are connected with the identification sequence of NheI.
DNA fragmentation between the MluI of carrier pGL3-Basic and NheI identification sequence is replaced with into CMRP-632, obtains weight
The recombinant vector is named as pGL3-CMRP-632 by group carrier, and in pGL3-CMRP-632, the 5 ' ends of CMRP-632 are with MluI's
Identify that sequence is connected, 3 ' ends are connected with the identification sequence of NheI.
DNA fragmentation between the MluI of carrier pGL3-Basic and NheI identification sequence is replaced with into CMRP-654, obtains weight
The recombinant vector is named as pGL3-CMRP-654 by group carrier, and in pGL3-CMRP-654, the 5 ' ends of CMRP-654 are with MluI's
Identify that sequence is connected, 3 ' ends are connected with the identification sequence of NheI.
DNA fragmentation between the MluI of carrier pGL3-Basic and NheI identification sequence is replaced with into CMRP-675, obtains weight
The recombinant vector is named as pGL3-CMRP-675 by group carrier, and in pGL3-CMRP-675, the 5 ' ends of CMRP-675 are with MluI's
Identify that sequence is connected, 3 ' ends are connected with the identification sequence of NheI.
DNA fragmentation between the MluI of carrier pGL3-Basic and NheI identification sequence is replaced with into CMRP-789, obtains weight
The recombinant vector is named as pGL3-CMRP-789 by group carrier, and in pGL3-CMRP-789, the 5 ' ends of CMRP-789 are with MluI's
Identify that sequence is connected, 3 ' ends are connected with the identification sequence of NheI.
DNA fragmentation between the MluI of carrier pGL3-Basic and NheI identification sequence is replaced with into CMRP-819, obtains weight
The recombinant vector is named as pGL3-CMRP-819 by group carrier, and in pGL3-CMRP-819, the 5 ' ends of CMRP-819 are with MluI's
Identify that sequence is connected, 3 ' ends are connected with the identification sequence of NheI.
DNA fragmentation between the MluI of carrier pGL3-Basic and NheI identification sequence is replaced with into CMRP-1051, obtains weight
The recombinant vector is named as pGL3-CMRP-1051 by group carrier, in pGL3-CMRP-1051, the 5 ' ends of CMRP-1051 with
The identification sequence of MluI is connected, and 3 ' ends are connected with the identification sequence of NheI.
2, CMRP has promoter activity in Neonatal Mouse cardiac muscle cell
2.1 cell transfecting
The recombinant vector of step 1 and pGL3-Basic are transfected respectively into Neonatal Mouse cardiac muscle cell.
2.1.1 the preparation of Neonatal Mouse cardiac muscle cell
1) 0.1% pancreatin is added in the HANKS buffer without calcium and 0.08% clostridiopetidase A is configured to digestive juice, use
0.05 μm of membrane filtration degerming.
2) Neonatal Mouse (tieing up experimental animal Co., Ltd of tonneau China in Beijing) heart is taken, it is slow that a certain amount of HANKS is added
Fliud flushing is shredded in capsule with scissors, organizes block size in diameter 1mm or so.
3) tissue block shredded and HANKS buffer are transferred in digestion bottle together, stand 1min, to tissue block whole
Abandoning HANKS buffer is inhaled after sinking to bottom of bottle.
4) 5ml digestive juice is added, the stirring digestion 6min in 37 C water baths takes out digestion bottle, stands 1min, inhales and abandon
Digestive juice.
5) 5ml digestive juice is added, the stirring digestion 6min in 37 C water baths takes out digestion bottle, stands 1min, inhales
Digestive juice is transferred in clean 15ml centrifuge tube out, is added in 5ml DMEM 10%FBS culture medium and digestive juice.Repeating should
Step 6 time.
6) by the digestive juice 1000r/min rationality 5min after neutralization, 1ml DMEM 10%FBS culture is added after abandoning supernatant
Base weight is outstanding.
7) it collects and repeatedly digests resulting re-suspension liquid, be filtered the tissue for removing and not digesting completely with the strainer of 50 mesh
Filtered cell re-suspension liquid is laid in 10ml culture dish by block, and 5ml DMEM 10%FBS culture medium is added, shakes up postposition
The 2h in 37 degrees Celsius of incubators, removes fibroblast in the way of differential velocity adherent, obtains Neonatal Mouse cardiac muscle cell's (heart
Myocyte).
2.1.2 cell transfecting
Specific steps are as follows:
1) opti-MEM culture medium is preheated in advance to 37 DEG C；Half an hour is by containing in step 2.1.1 culture dish before transfecting
There is the media transfer of Neonatal Mouse cardiac muscle cell into 50ml centrifuge tube, and culture medium is changed into the opti-MEM of preheating.
2) recombinant vector of step 1 or pGL3-Basic and lipo2000 are separately added into the opti-MEM of certain volume
In, the ratio of carrier and lipo2000 are 1 μ g:2 μ l, and room temperature is incubated for 5min.
3) opti-MEM containing lipo2000 is added in the opti-MEM containing plasmid, room temperature is incubated for after mixing
30min。
4) the opti-MEM solution mixed is added in the cultivating system of step 1), after 6h more by opti-MEM culture medium
It is changed to DMEM 10%FBS culture medium.
Finally obtain respectively by pGL3-CMRP, pGL3-CMRP-533, pGL3-CMRP-555, pGL3-CMRP-575,
pGL3-CMRP-589、pGL-3CMRP-614、pGL3-CMRP-632、pGL3-CMRP-654、pGL3-CMRP-675、pGL3-
CMRP-789, pGL3-CMRP-819, pGL3-CMRP-1051 and pGL3-Basic import cardiac muscle cell and obtain recombinant cell, will
These recombinant cells are successively named as C-pGL3-CMRP, C-pGL3-CMRP-533, C-pGL3-CMRP-555, C-pGL3-
CMRP-575、C-pGL3-CMRP-589、C-pGL3-CMRP-614、C-pGL3-CMRP-632、C-pGL3-CMRP-654、C-
PGL3-CMRP-675, C-pGL3-CMRP-789, C-pGL3-CMRP-819, C-pGL3-CMRP-1051 and C-pGL3-Basic.
The measurement of 2.2 relative luciferase activities and analysis
It is carried out using double Fluorescence kits of promega, the specific steps are as follows:
1) the Passive Lysis buffer (PLB) of 1 times of volume is added in 4 times of volume ultrapure waters, is uniformly mixed.
2) cell culture medium after step 2.1.2 transfection is inhaled and is abandoned, the PLB diluted is added, in shaking cracking on shaking table
15min。
3) lysate suction is transferred in centrifuge tube, wink is from 1min.
4) it takes 10 μ l of supernatant to be added in a hole of 96 orifice plates, 50 μ l LARII reagents is added, react the several seconds, utilize enzyme mark
Instrument detects firefly luciferase activity.
5) 50 μ lStop&Gloi Reagent are added, react the several seconds, utilize microplate reader detection renilla luciferase activity.
Luciferase of the firefly luciferase plain (Luc) of each recombinant cell relative to renilla luciferase vigor (Rluc)
The testing result of relative activity is as shown in Figure 1.
The results show that C-pGL3-CMRP, C-pGL3-CMRP-575, C-pGL3-CMRP-589, C-pGL3-CMRP-614,
C-pGL3-CMRP-632、C-pGL3-CMRP-654、C-pGL3-CMRP-675、C-pGL3-CMRP-789、C-pGL3-CMRP-
The relative luciferase activity of 819 and C-pGL3-CMRP-1051 it is extremely significant be higher than C-pGL3-Basic, show CMRP,
CMRP-575, CMRP-589, CMRP-614, CMRP-632, CMRP-654, CMRP-675, CMRP-789, CMRP-819 and
CMRP-1051 all has promoter activity in cardiac muscle cell；The fluorescence of C-pGL3-CMRP-555 and C-pGL3-CMRP-533
Plain enzyme relative activity is respectively 20% of C-pGL3-CMRP or so and 10% or so, the luciferase phase of C-pGL3-CMRP-533
To activity and C-pGL3-Basic without significant difference, show that the promoter of CMRP-555 and CMRP-533 in cardiac muscle cell is living
Property reduce, CMRP-555 and CMRP-533 are in cardiac muscle cell almost without promoter activity.
3, CMRP promoter activity specific detection
By pGL3-CMRP, pGL3-CMRP-575, pGL3-CMRP-589, pGL3-CMRP-614, pGL3- of step 1
CMRP-632、pGL3-CMRP-654、pGL3-CMRP-675、pGL3-CMRP-789、pGL3-CMRP-819、pGL3-CMRP-
1051 and pGL3-Basic transfects Hek293 cell (ATCC cell bank, number CRL-1573) and Hela cell respectively, and (ATCC is thin
Born of the same parents library, number CCL-2.1), the specific method is as follows:
1) opti-MEM culture medium is preheated in advance to 37 DEG C；It is thin with corresponding culture medium culture Hek293 cell or Hela
Born of the same parents, and culture medium is changed into the opti-MEM of preheating.
2) carrier and lipo2000 are separately added into the opti-MEM of certain volume, the ratio of carrier and lipo2000 are
1 μ g:2 μ l, room temperature are incubated for 5min.
3) opti-MEM containing lipo2000 is added in the opti-MEM containing carrier, room temperature is incubated for after mixing
30min。
4) the opti-MEM solution mixed is added in the cultivating system of step 1), after 6h more by opti-MEM culture medium
It is changed to the culture medium of corresponding cell.
Wherein, the culture medium of Hek293 cell is DMEM (gibco)+10%FBS (thermo fisher), Hela cell
Culture medium be DMEM (gibco)+10%FBS (thermo fisher).
By pGL3-CMRP, pGL3-CMRP-575, pGL3-CMRP-589, pGL3-CMRP-614, pGL3-CMRP-632,
PGL3-CMRP-654, pGL3-CMRP-675, pGL3-CMRP-789, pGL3-CMRP-819, pGL3-CMRP-1051 and pGL3-
Basic import Hek293 cell obtain recombinant cell, by these recombinant cells be successively named as Hek293-pGL3-CMRP,
Hek293-pGL3-CMRP-575、Hek293-pGL3-CMRP-589、Hek293-pGL3-CMRP-614、Hek293-pGL3-
CMRP-632、Hek293-pGL3-CMRP-654、Hek293-pGL3-CMRP-675、Hek293-pGL3-CMRP-789、
Hek293-pGL3-CMRP-819, Hek293-pGL3-CMRP-1051 and Hek293-pGL3-Basic；
By pGL3-CMRP, pGL3-CMRP-575, pGL3-CMRP-589, pGL3-CMRP-614, pGL3-CMRP-632,
PGL3-CMRP-654, pGL3-CMRP-675, pGL3-CMRP-789, pGL3-CMRP-819, pGL3-CMRP-1051 and pGL3-
Basic imports Hela cell and obtains recombinant cell, these recombinant cells are successively named as Hela-pGL3-CMRP, Hela-
pGL3-CMRP-575、Hela-pGL3-CMRP-589、Hela-pGL3-CMRP-614、Hela-pGL3-CMRP-632、Hela-
pGL3-CMRP-654、Hela-pGL3-CMRP-675、Hela-pGL3-CMRP-789、Hela-pGL3-CMRP-819、Hela-
PGL3-CMRP-1051 and Hela-pGL3-Basic.
Analyze the fluorescence of above-mentioned each recombinant cell respectively with analysis method according to relative luciferase activity measurement in step 2
Plain enzyme relative activity, the results show that Hek293-pGL3-CMRP, Hek293-pGL3-CMRP-575, Hek293-pGL3-CMRP-
589、Hek293-pGL3-CMRP-614、Hek293-pGL3-CMRP-632、Hek293-pGL3-CMRP-654、Hek293-
PGL3-CMRP-675, Hek293-pGL3-CMRP-789, Hek293-pGL3-CMRP-819 and Hek293-pGL3-CMRP-
1051 relative luciferase activity is and Hek293-pGL3-Basic is without significant difference；Hela-pGL3-CMRP,Hela-
pGL3-CMRP-575、Hela-pGL3-CMRP-589、Hela-pGL3-CMRP-614、Hela-pGL3-CMRP-632、Hela-
PGL3-CMRP-654, Hela-pGL3-CMRP-675, Hela-pGL3-CMRP-789, Hela-pGL3-CMRP-819 and Hela-
The relative luciferase activity of pGL3-CMRP-1051 is and Hela-pGL3-Basic is without significant difference；Show CMRP, CMRP-
575, CMRP-589, CMRP-614, CMRP-632, CMRP-654, CMRP-675, CMRP-789, CMRP-819 and CMRP-1051
Do not have promoter activity in Hek293 cell and Hela cell.C-pGL3-CMRP, Hek293-pGL3-CMRP with
The relative luciferase activity of Hela-pGL3-CMRP is as shown in Figure 2.
The above result shows that CMRP, CMRP-575, CMRP-589, CMRP-614, CMRP-632, CMRP-654, CMRP-
675, the promoter activity of CMRP-789, CMRP-819 and CMRP-1051 all have cardiac myocytespecific.
Claims (8)
1.DNA molecule is 3 ' 927-1501 nucleotide sequences of the end at least containing sequence 1 in ordered list, and from sequence 1
The 927th start, extend according to the nucleotide sequence of sequence 1 to 5 ' ends of sequence 1, obtaining length is 575 to 1501bp's
Any one DNA fragmentation；The DNA molecular has promoter function；
Last nucleotide of the DNA molecular is the 1501st of sequence 1.
2. DNA molecular according to claim 1, it is characterised in that: the DNA molecular is following A 1)-A8) in any
Kind DNA fragmentation:
A1) 3 ' 913-1501 nucleotide sequences of the end at least containing sequence 1 in ordered list of nucleotide sequence, and from sequence
The 913rd of column 1 starts, extends according to the nucleotide sequence of sequence 1 to 5 ' ends of sequence 1, and obtaining length is 589 to 1501bp
Any one DNA fragmentation；
A2) 3 ' 888-1501 nucleotide sequences of the end at least containing sequence 1 in ordered list of nucleotide sequence, and from sequence
The 888th of column 1 starts, extends according to the nucleotide sequence of sequence 1 to 5 ' ends of sequence 1, and obtaining length is 614 to 1501bp
Any one DNA fragmentation；
A3) 3 ' 870-1501 nucleotide sequences of the end at least containing sequence 1 in ordered list of nucleotide sequence, and from sequence
The 870th of column 1 starts, extends according to the nucleotide sequence of sequence 1 to 5 ' ends of sequence 1, and obtaining length is 632 to 1501bp
Any one DNA fragmentation；
A4) 3 ' 848-1501 nucleotide sequences of the end at least containing sequence 1 in ordered list of nucleotide sequence, and from sequence
The 848th of column 1 starts, extends according to the nucleotide sequence of sequence 1 to 5 ' ends of sequence 1, and obtaining length is 654 to 1501bp
Any one DNA fragmentation；
A5) 3 ' 827-1501 nucleotide sequences of the end at least containing sequence 1 in ordered list of nucleotide sequence, and from sequence
The 827th of column 1 starts, extends according to the nucleotide sequence of sequence 1 to 5 ' ends of sequence 1, and obtaining length is 675 to 1501bp
Any one DNA fragmentation；
A6) 3 ' 713-1501 nucleotide sequences of the end at least containing sequence 1 in ordered list of nucleotide sequence, and from sequence
The 713rd of column 1 starts, extends according to the nucleotide sequence of sequence 1 to 5 ' ends of sequence 1, and obtaining length is 789 to 1501bp
Any one DNA fragmentation；
A7) 3 ' 683-1501 nucleotide sequences of the end at least containing sequence 1 in ordered list of nucleotide sequence, and from sequence
The 683rd of column 1 starts, extends according to the nucleotide sequence of sequence 1 to 5 ' ends of sequence 1, and obtaining length is 819 to 1501bp
Any one DNA fragmentation；
A8) 3 ' 451-1501 nucleotide sequences of the end at least containing sequence 1 in ordered list of nucleotide sequence, and from sequence
The 451st of column 1 start, extends according to the nucleotide sequence of sequence 1 to 5 ' ends of sequence 1, obtain length be 1051 to
Any one DNA fragmentation of 1501bp.
3. DNA molecular according to claim 2, it is characterised in that: the DNA molecular is following 1) -10) in it is any one
Kind:
1) DNA molecular shown in 927-1501 nucleotide of sequence 1 in sequence table；
2) DNA molecular shown in 913-1501 nucleotide of sequence 1 in sequence table；
3) DNA molecular shown in 888-1501 nucleotide of sequence 1 in sequence table；
4) DNA molecular shown in 870-1501 nucleotide of sequence 1 in sequence table；
5) DNA molecular shown in 848-1501 nucleotide of sequence 1 in sequence table；
6) DNA molecular shown in 827-1501 nucleotide of sequence 1 in sequence table；
7) DNA molecular shown in 713-1501 nucleotide of sequence 1 in sequence table；
8) DNA molecular shown in 683-1501 nucleotide of sequence 1 in sequence table；
9) DNA molecular shown in 451-1501 nucleotide of sequence 1 in sequence table；
10) DNA molecular shown in 1-1501 nucleotide of sequence 1 in sequence table.
4. it is following B1 the biomaterial containing any DNA molecular of claim 1-3) any one of to B13):
B1 the expression cassette) containing any DNA molecular of claim 1-3；
B2 the recombinant vector) containing any DNA molecular of claim 1-3；
B3) contain B1) recombinant vector of the expression cassette；
B4 the recombinant microorganism) containing any DNA molecular of claim 1-3；
B5) contain B1) recombinant microorganism of the expression cassette；
B6) contain B2) recombinant microorganism of the recombinant vector；
B7) contain B3) recombinant microorganism of the recombinant vector；
B8 the transgenetic animal cell system) containing any DNA molecular of claim 1-3；
B9) contain B1) the transgenetic animal cell system of the expression cassette；
B10) the transgenic animals tissue containing any DNA molecular of claim 1-3；
B11) contain B1) the transgenic animals tissue of the expression cassette；
B12 the transgenic animal organ) containing any DNA molecular of claim 1-3；
B13) contain B1) transgenic animal organ of the expression cassette.
5. any application in following M1-M3:
Any DNA molecular of M1, claim 1-3 is as the application in specific heart promoter；
Any DNA molecular of M2, claim 1-3 is as the application in myocardium specific promoter；
Any DNA molecular of M3, claim 1-3 is as the application in cardiac muscle cell's specific promoter；
The application is the application of non-disease diagnosing and treating.
6. any application in following N1-N3:
Any DNA molecular of N1, claim 1-3 starts the application in destination gene expression in heart；
Any DNA molecular of N2, claim 1-3 starts the application in destination gene expression in cardiac muscle；
Any DNA molecular of N3, claim 1-3 starts the application in destination gene expression in cardiac muscle cell；
The application is the application of non-disease diagnosing and treating.
7. any DNA molecular of claim 1-3 starts the application in destination gene expression in animal；It is described to be expressed as the heart
Dirty specifically expressing or cardiac muscle cell's specifically expressing；The application is the application of non-disease diagnosing and treating.
8. any DNA molecular of claim 1-3 is cultivating the application in transgenic animals.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610374248.3A CN105779457B (en) | 2016-05-31 | 2016-05-31 | Cardiac muscle cell's specific promoter and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610374248.3A CN105779457B (en) | 2016-05-31 | 2016-05-31 | Cardiac muscle cell's specific promoter and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105779457A CN105779457A (en) | 2016-07-20 |
| CN105779457B true CN105779457B (en) | 2019-02-12 |
Family
ID=56375434
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610374248.3A Active CN105779457B (en) | 2016-05-31 | 2016-05-31 | Cardiac muscle cell's specific promoter and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105779457B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1483040A (en) * | 2000-12-07 | 2004-03-17 | ��������I�����ɷ� | Heart ankyrin repeat protein gene upstream sequence, carrier containing the same sequence and usage thereof |
| CN101985628A (en) * | 2010-08-13 | 2011-03-16 | 哈尔滨医科大学 | Method for building special microRNA knock-down mouse model of heart |
| CN103173451A (en) * | 2013-04-15 | 2013-06-26 | 江苏省人民医院 | Myocardial specific promoter |
-
2016
- 2016-05-31 CN CN201610374248.3A patent/CN105779457B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1483040A (en) * | 2000-12-07 | 2004-03-17 | ��������I�����ɷ� | Heart ankyrin repeat protein gene upstream sequence, carrier containing the same sequence and usage thereof |
| CN101985628A (en) * | 2010-08-13 | 2011-03-16 | 哈尔滨医科大学 | Method for building special microRNA knock-down mouse model of heart |
| CN103173451A (en) * | 2013-04-15 | 2013-06-26 | 江苏省人民医院 | Myocardial specific promoter |
Non-Patent Citations (4)
| Title |
|---|
| Morphogenesis of T-tubules in heart cells: the role of junctophilin-2;HAN Jing et al.;《SCIENCE CHINA》;20130731;第56卷(第7期);647-652 |
| XM006235526.2;NCBI;《GenBank》;20140807;全文 |
| 人心肌肌球蛋白轻链基因启动子驱动的GFP和Luc报告基因在人心肌细胞系中的表达;许秀芳 等;《中国病理生理杂志》;20121231;第28卷(第9期);1722-1728 |
| 心肌细胞横管的形态发生：Junctophilin-2的作用;韩晶 等;《中国科学：生命科学》;20131231;第43卷(第9期);738-744 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105779457A (en) | 2016-07-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Millay et al. | Structure–function analysis of myomaker domains required for myoblast fusion | |
| Stühmer et al. | Ectopic expression of the Dlx genes induces glutamic acid decarboxylase and Dlx expression | |
| JP7275043B2 (en) | Enhanced hAT Family Transposon-Mediated Gene Transfer and Related Compositions, Systems and Methods | |
| CN108504657A (en) | The method for knocking out HEK293T cell KDM2A genes using CRISPR-CAS9 technologies | |
| Gumbs et al. | Characterization of HIV-1 infection in microglia-containing human cerebral organoids | |
| Lin et al. | Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning | |
| US11278570B2 (en) | Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems, and methods | |
| Adame et al. | An undergraduate laboratory class using CRISPR/Cas9 technology to mutate drosophila genes | |
| Vogt et al. | Viral-mediated labeling and transplantation of medial ganglionic eminence (MGE) cells for in vivo studies | |
| Rappleye et al. | Opto-MASS: a high-throughput engineering platform for genetically encoded fluorescent sensors enabling all-optical in vivo detection of monoamines and opioids | |
| Klein et al. | Isolation of endocardial and coronary endothelial cells from the ventricular free wall of the rat heart | |
| CN107760707A (en) | A kind of foundation for the self-activation Gal4/UAS system expression boxes for strengthening gene expression | |
| Kari et al. | Embryo microinjection and electroporation in the chordate Ciona intestinalis | |
| CN105779457B (en) | Cardiac muscle cell's specific promoter and its application | |
| Zhang et al. | Gene knock-in by CRISPR/Cas9 and cell sorting in macrophage and T cell lines | |
| Zyrianova et al. | Isolation of myoepithelial cells from adult murine lacrimal and submandibular glands | |
| CN104711240B (en) | The application of Avianreovirus σ A albumen and its relevant biological material | |
| Fuster-García et al. | Application of CRISPR tools for variant interpretation and disease modeling in inherited retinal dystrophies | |
| CN103215267B (en) | Suppress siRNA and its application of influenza virus related gene | |
| Đorđević et al. | Nucleofection as an efficient method for alpha Tc1-6 cell line transfection | |
| CN110408621A (en) | The methods and applications of macaque ROSA26 gene and its gene modification | |
| Henley et al. | Reliable identification of living dopaminergic neurons in midbrain cultures using RNA sequencing and TH-promoter-driven eGFP expression | |
| CN104762274B (en) | The application of Avianreovirus σ NS albumen and its relevant biological material | |
| WO2020223240A1 (en) | Affinity-based multiplexing for live-cell monitoring of complex cell populations | |
| Svoboda | On board a raft or boat in the retrovirus sea |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |