CN110117618A - A kind of labeling method of Sepiella maindroni archaeocyte - Google Patents
A kind of labeling method of Sepiella maindroni archaeocyte Download PDFInfo
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Abstract
The present invention provides a kind of labeling method of Sepiella maindroni archaeocyte, belongs to gene-tagging techniques field, including, collect the Sepiella maindroni fertilized eggs of 1- cell stage；Double digestion is inserted into 3 ' the UTR fragment products of amplification Vasa and/or EGFP gene amplified production of PGEM-T easy carrier, connection；Products therefrom Sal I enzyme linearization process will be connected, and synthesizes 3 ' UTR mRNA of Vasa in vitro；3 ' UTR mRNA of gained Vasa is injected in fertilized eggs to be marked.Labeling method of the present invention merges 3 ' the UTR mRNA of Vasa of Vasa gene chemical synthesis to early stage fertilized eggs by injection GFP, make GFP in PGCs specifically expressing so that PGCs is marked, in vivo marker can be carried out to the Sepiella maindroni archaeocyte before separation freezing, accuracy is high, technology is easy, and the period, short purposes was wide.
Description
Technical field
The invention belongs to gene-tagging techniques fields, and in particular to a kind of label of Sepiella maindroni archaeocyte
Method.
Background technique
Siphonopods belongs to mollusk, is the important component in marine ecosystems, while balancing to marine ecology
There is indispensable role.Cuttlefish belongs to Mollusca (Mollusca), Cephalopoda (Cephalopoda), widely distributed
In each sea area, occupy an important position in biosystem evolution.Its delicious meat is palatable, has good nutritive value,
Protein content is high.Other than edible, there are also very high medical value, ink sac can promote blood clotting and have
Bacteria resistance function, cuttlebone can be medicinal, and otolith can be used to analyze the age of species and the change in time and space of marine environment.In addition, black
Highly important biotic factor in crafty or marine ecosystems, plays highly important angle in the food chain of the ecosystem
Color plays very important effect to the ecological balance for maintaining marine ecosystem.In recent years, cuttlefish is as important economic cephalopodium
Class plays a key role in marine ecosystems.Due to overfishing and environmental change, stock number is once by very big
It destroys, natural resources obviously fails, and yield sharply declines, and fishing season disappears, and closes in imminent danger.Since the last century 80's,
Have benefited from the breakthrough of artificial breeding and Feeding Technique, stock number is in gradually recovery.
Reproduction cell is just separated with body cell in embryonic development early stage, is transmitted hereditary information by being divided into both sexes gamete
To the next generation, multiply species from generation to generation, life and growth in nature.Archaeocyte PGCs is the reproduction cell occurred earliest, it is in embryo
The early formation that fetal hair is educated.PGCs is divided into spermatogonium and oogonium respectively and finally generates in original sexual gland atomization
Sperm and ovum.Because PGCs has the potential for being divided into both sexes gamete, by the way that the offspring of donor can be generated after heteroplastic transplantation, because
This, PGCs is not only the ideal material of research cell differentiation and migration, and by the research to PGCs genesis and development, for life
Growing development basic theory, Germ-plasma resources protection all has far-reaching significance, and thus it is raw to have become marine organisms breeding field by PGCs
The research hotspot of cell colonization operation.However position and quantity of the fish PGCs in embryo development procedure are still unclear at present,
Significantly limit the development of PGCs freezen protective technology.Embryo's whole mount in situ hybridization can track PGCs in embryo development procedure
Migration with regard to variation of quantity, but this technology mainly easily goes in the fish of egg membrane to apply in fresh-water fishes, and traditional embryo is whole former
Sampling and preserving type are not particularly suited for fish embryo samples, while complex steps before the hybridization of position.Therefore, Mans needleless is established
The positioning and marking method of PGCs in cuttlefish embryo development procedure discloses the origin Sepiella maindroni PGCs and migration model, is graceful
Preservation, genital regulating mechanism and the artificial propagation breeding of family name's sepiella maindroni de Rochebrune germ plasm resource provide necessary rudimentary knowledge.
Summary of the invention
The purpose of the present invention is to provide it is a kind of can to the Sepiella maindroni archaeocyte before separation freezing into
Row in vivo marker, accuracy is high, and technology is easy, the label side of the wide Sepiella maindroni archaeocyte of period short purposes
Method.
The technical solution that the present invention is taken to achieve the above object are as follows:
A kind of labeling method of Sepiella maindroni archaeocyte, uses Vasa gene for marker gene, including,
Collect the Sepiella maindroni fertilized eggs of 1- cell stage；
Double digestion is inserted into 3 ' the UTR fragment products of amplification Vasa of PGEM-T easy carrier and/or EGFP gene amplification produces
Object, connection；
Products therefrom Sal I enzyme linearization process will be connected, and synthesizes 3 ' UTR mRNA of Vasa in vitro；
3 ' UTR mRNA of gained Vasa is injected in fertilized eggs to be marked.
Vasa gene is a kind of source of parents effector and has important role to the migration and differentiation of reproduction cell, can be adjusted
The translation for controlling protein regulates and controls other protein by mRNA and positions in cytoplasm and with other interactions between protein etc., therefore
Vasa is taken as system genitale specific molecular marker and is widely used in studying the origin PGCs, migration, differentiation etc.；3 ' UTR of Vasa can
Stability of the chimera mRNA in PGCs is maintained, and this function is relatively conservative in evolution, labeling method of the present invention is logical
It crosses in 3 ' the UTR mRNA to early stage fertilized eggs of Vasa of injection GFP fusion Vasa gene chemical synthesis, makes GFP in the PGCs of embryo
So that PGCs is marked, the Sepiella maindroni archaeocyte before capable of freezing to separation carries out middle specifically expressing
In vivo marker, accuracy is high, provides the foundation for PGCs separation freezing, technology is easy, and the period, short purposes was wide.
Preferably, Vasa gene is selected from zebra fish Vasa, Tilapia mossambica Vasa, turbot Vasa, carp Vasa or green Medaka
Vasa。
Preferably, 3 ' UTR fragment amplification primer of Vasa are as follows:
Vasa-F:5 '-GCTGCTGATGTCTAATGCGATT-3 '；
Vasa-R:5 '-ATCAGCTATTTAATTATCGGAGATC-3 '.
Preferably, 3 ' UTR fragment amplification step of Vasa are as follows:
Genome total serum IgE is extracted, reverse transcription synthesizes cDNA, carries out PCR expansion with primer using 3 ' UTR fragment amplification of Vasa
Increase；The wherein response procedures of PCR amplification are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 70s, 30 circulations；Most
72 DEG C of extension 10min afterwards.
More preferably, hydroxychloroquine sulfate is added to 1-2 μM after tissue sample grinding during Total RNAs extraction, then exist
Frequency 2-4kHz, intensity 12-15mW/cm2Ultrasonic wave under carry out ultrasonic wave circulation stimulation after be centrifuged again.Under above-mentioned ultrasonic wave
Stimulation under, occur that membrane permeability can be changed in the ultrasonic wave vortex of cell edges and surrounding liquid, under above-mentioned ultrasonic wave
Stimulation under, occur that membrane permeability can be changed in the ultrasonic wave vortex of cell edges and surrounding liquid, sufficiently release is intracellular
RNA；Simultaneously because the cell membrane surface electrochemistry disorder that ultrasonic wave generates can adsorb hydroxychloroquine sulfate in cell membrane surface, make
It obtains hydroxychloroquine sulfate and gives full play to and inhibit the active effect of lysosome, guarantee the integrality of RNA, improve expanding effect, finally mention
The accuracy of high labeling method.
Preferably, EGFP gene amplification primers are as follows:
EGFP-F:5 '-TAGCTGACGTCGCCCCATGGTTAGC-3 '；
EGFP-R:5 '-TATGTCGCGGTTACTTGCACAGTCGTC-3 '.
Preferably, introducing Aat II and Sac II restriction enzyme site in EGFP gene amplified production.
Preferably, being diluted to 100ng/ μ l with DEPC water, 0.02% phenol red solution before 3 ' UTR mRNA injection.
Preferably, the external synthesis of 3 ' UTR mRNA of Vasa is carried out using the external synthetic agent box of RNA；Vasa 3'
The step of external synthesis of UTR mRNA are as follows: after cultivating 1.5-2.5h during 35-40 DEG C, 80-130rpm magnetic agitation, mend
Add the MgCl of 80-100 μ L 1M2With the RNA Ⅲ of 15-22 μ L 0.3mM, sustained response 3.5-4.5h.MgCl2And ribose
Nuclease III can well outside control volume in synthesis process under the conditions of magnetic agitation of the present invention transcription system and digestion body
System, so that the RNA product 5 ' that generation is transcribed in vitro is held and 3 ' there will be no additional increased nucleotide, external turn for improving RNA
Record completeness and purpose RNA amount obtain transcription amount height, the higher 3 ' UTR of purpose Vasa of purity convenient for separation and purifying
MRNA, the final accuracy for improving labeling method of the present invention.
Preferably, the fertilized eggs after label are cultivated in embryo medium in 25-30 DEG C, in fluorescence microscopy microscopic observation
Different development stage fluorescent protein expression situation.
Compared with prior art, the invention has the benefit that
Labeling method of the present invention by injection GFP merge Vasa gene chemical synthesis 3 ' UTR mRNA of Vasa to early stage fertilization
In ovum, making GFP, specifically expressing, can be to the Mans before separation freezing so that PGCs is marked in the PGCs of embryo
Sepiella maindroni de Rochebrune archaeocyte carries out in vivo marker, and accuracy is high, provides the foundation for PGCs separation freezing, and technology is easy,
Period, short purposes was wide；The method of the present invention can be improved the in-vitro transcription completeness and purpose RNA amount of RNA, convenient for separation and purify,
Obtain transcription amount height, higher 3 ' the UTR mRNA of purpose Vasa of purity, the final accuracy for improving labeling method of the present invention.
Present invention employs above-mentioned technical proposals to provide a kind of labeling method of Sepiella maindroni archaeocyte, more
The deficiencies in the prior art, reasonable design, easy operation are mended.
Detailed description of the invention
Fig. 1 is that purpose RNA amount obtained by the external synthesis of 3 ' UTR mRNA of Vasa accounts for transcription gained total serum IgE amount in embodiment 4
Percentage result.
Specific embodiment
In the following, in conjunction with specific embodiments to a kind of Sepiella maindroni archaeocyte of an embodiment of the present invention
Labeling method is described further.
Embodiment 1:
A kind of labeling method of Sepiella maindroni archaeocyte, uses turbot Vasa gene for marker gene,
Including,
1) fertilized eggs are collected:
By Sepiella maindroni raising water temperature be 26 DEG C, periodicity of illumination be 14h (illumination): under conditions of 10h (dark)
It uses, when dusk separates male and female Sepiella maindroni, and second day early morning mixed male and female according to 1:1 ratio, wait produce
Ovum finishes the Sepiella maindroni fertilized eggs for collecting 1- cell stage；
2) extract turbot genome total serum IgE, reverse transcription synthesize cDNA, using 3 ' UTR fragment amplification primer of Vasa into
Row PCR amplification；The wherein response procedures of PCR amplification are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 70s, 30
Circulation；Last 72 DEG C of extensions 10min；
Wherein, the step of extraction turbot genome total serum IgE, reverse transcription synthesizes cDNA are as follows:
A. the 2ml EP of no RNA enzyme is taken to manage, 500ul Trizol, the tweezers clamping group steeped with alcohol is added in pipette tips
Tissue samples are dissolved in Trizol liquid, and electric homogenizer grinding is dissolved completely in liquid until tissue, are filled Trizol and are arrived
Hydroxychloroquine sulfate is added to 1-2 μM, then in frequency 2-4kHz, intensity 12-15mW/cm in 1mL2Ultrasonic wave under carry out ultrasound
Wave circulation stimulation, 4 DEG C, 12000rpm, 10min centrifugation；Under the stimulation under above-mentioned ultrasonic wave, occur cell edges and around
The ultrasonic wave vortex of liquid can change membrane permeability, under the stimulation under above-mentioned ultrasonic wave, occur cell edges and around
The ultrasonic wave vortex of liquid can change membrane permeability, sufficiently release intracellular rna；Simultaneously because the cell membrane that ultrasonic wave generates
Surface electrochemistry disorder can adsorb hydroxychloroquine sulfate in cell membrane surface, so that hydroxychloroquine sulfate gives full play to inhibition lysosome
Active effect guarantees the integrality of RNA, improves expanding effect, the final accuracy for improving labeling method；
It takes supernatant to put into 1.5mL EP pipe, impurity cannot be drawn onto；
The chloroform of 200ul is added, acutely shakes, is stored at room temperature 5 minutes, 4 DEG C, 12000rpm, 15min are centrifuged；
It takes top layer's liquid in three layers into new 1.5mL EP pipe, 500ul isopropanol, room temperature 10min or -20 is added
DEG C overnight；
4 DEG C, 12000rpm, 10min centrifugation remove supernatant, it is seen that white plates, that is, RNA；
75% ethyl alcohol of 1mL is added, pressure-vaccum mixes, and stands 5min；
4 DEG C, 7500rpm, 5min centrifugation remove supernatant, the dry 10min of draught cupboard；
20ul DEPC water, 4 DEG C of overnight or -20 DEG C of preservations are added；
The inspection of RNA mass: be dissolved in DEPC processing water RNA using nucleic acid-protein detector measure its 260nm with
And the absorbing wavelength at 280nm, according to relevant information, 260/280 value shown between 1.8~2.0 extracted RNA mass compared with
Pure, the foreign protein and polysaccharose substance content contained is less, lower than this range RNA it is not recommended that use, illustrate higher than 2.0
Extracted RNA has more degradation, is not suitable for the experiment of next step amplification gene full-length cDNA and uses.After the completion of measurement, 2~3ml is taken
It is splined on 1% agarose gel electrophoresis, deposition condition is set as 135V and 150mA, electrophoresis 15min, in gel image analyser
Middle observation RNA electrophoretic band extracts the preferable RNA of quality and is able to observe that apparent 28S and 18S band, and the ratio between brightness is 2:
1；
B.cDNA synthesis:
Using M-MLV reverse transcription reagent box, specific steps are as follows:
Following reaction system is added in 0.2ml centrifuge tube:
Total serum IgE 5.0ul
Oligo DT 1.0ul
It is centrifuged, is placed in PCR instrument after mixing, setting temperature is 70 DEG C, is taken out immediately after reacting 10min, ice bath 2min
Terminate.Following reagent is continuously added in above-mentioned reaction system:
Be centrifuged after mixing, in setting reaction condition in PCR instrument as 42 DEG C of 60min, 70 DEG C of 15min, after take out rapidly
Ice bath 15min, extremely -20 DEG C of preservation spare, and long-term preservation is then placed in -80 DEG C of refrigerators；
3) external structure of 3 ' UTR mRNA of Vasa:
A. design primer expands 3 ' UTR segment of Vasa, is inserted into PGEM-T easy carrier, and verify and be correctly inserted into direction；
B. design primer expands EGFP gene, and introduces Aat II and Sac II restriction enzyme site, double digestion amplified production and
The product that step A is obtained, connection；
C. the product obtained with Sal I enzyme linearization step B, and 3 ' UTR mRNA are synthesized in vitro:
External synthesis is carried out using the external synthetic agent box of RNA；The step of external synthesis of 3 ' UTR mRNA of Vasa are as follows:
After cultivating 2.5h during 40 DEG C, 130rpm magnetic agitation, the MgCl of 100 μ L 1M is added2With the ribose core of 22 μ L 0.3mM
Sour enzyme III, sustained response 4.5h.MgCl2It can be closed outside control volume well under the conditions of the magnetic agitation with RNA Ⅲ
At transcription system and digestion system in the process, increase so that the end of RNA product 5 ' generated and 3 ' is transcribed in vitro there will be no additional
The nucleotide added improves the in-vitro transcription completeness and purpose RNA amount of RNA, and convenient for separation and purifying, it is high, pure to obtain transcription amount
Higher 3 ' UTR mRNA of purpose Vasa is spent, the final accuracy for improving labeling method.
4) microinjection:
3 ' UTR mRNA of gained Vasa DEPC water, 0.02% phenol red solution are diluted to 100ng/ μ l, are injected in fertilization
Ovum is marked, while the fertilized eggs after label are cultivated in embryo medium in 27 DEG C, different under the microscope in fluorescence microscopy
Developmental stage fluorescent protein expression situation.
Vasa gene is a kind of source of parents effector and has important role to the migration and differentiation of reproduction cell, can be adjusted
The translation for controlling protein regulates and controls other protein by mRNA and positions in cytoplasm and with other interactions between protein etc., therefore
Vasa is taken as system genitale specific molecular marker and is widely used in studying the origin PGCs, migration, differentiation etc.；3 ' UTR of Vasa can
Stability of the chimera mRNA in PGCs is maintained, and this function is relatively conservative in evolution, which passes through note
It penetrates in 3 ' the UTR mRNA to early stage fertilized eggs of Vasa of GFP fusion Vasa gene chemical synthesis, keeps GFP special in the PGCs of embryo
So that PGCs is marked, the Sepiella maindroni archaeocyte before capable of freezing to separation carries out living body for different expression
Label, accuracy is high, provides the foundation for PGCs separation freezing, technology is easy, and the period, short purposes was wide.
Above-mentioned 3 ' UTR fragment amplification primer of Vasa are as follows:
Vasa-F:5 '-GCTGCTGATGTCTAATGCGATT-3 '；
Vasa-R:5 '-ATCAGCTATTTAATTATCGGAGATC-3 '.
Above-mentioned EGFP gene amplification primers are as follows:
EGFP-F:5 '-TAGCTGACGTCGCCCCATGGTTAGC-3 '；
EGFP-R:5 '-TATGTCGCGGTTACTTGCACAGTCGTC-3 '.
Aat II and Sac II restriction enzyme site are introduced in above-mentioned EGFP gene amplified production.
100ng/ μ l is diluted to DEPC water, 0.02% phenol red solution before above-mentioned 3 ' UTR mRNA injection.
Embodiment 2:
A kind of labeling method of Sepiella maindroni archaeocyte, uses turbot Vasa gene for marker gene,
Including,
1) the Sepiella maindroni fertilized eggs of 1- cell stage are collected, the specific steps are the same as those in embodiment 1；
2) 3 ' the UTR fragment products of amplification Vasa of double digestion insertion PGEM-T easy carrier and/or EGFP gene amplification
Product, connection, the specific steps are the same as those in embodiment 1；
3) products therefrom Sal I enzyme linearization process will be connected, and synthesizes 3 ' UTR mRNA of Vasa in vitro, wherein body
Outer synthesis is carried out using the external synthetic agent box of RNA；The step of external synthesis of 3 ' UTR mRNA of Vasa are as follows: 38 DEG C,
After cultivating 2.0h during 100rpm magnetic agitation, the MgCl of 90 μ L 1M is added2With the RNA Ⅲ of 20 μ L 0.3mM,
Sustained response 4.0h；
4) 3 ' UTR mRNA of gained Vasa DEPC water, 0.02% phenol red solution are diluted to 100ng/ μ l, be injected in by
Smart ovum is marked, at the same mark after fertilized eggs in embryo medium in 27 DEG C cultivate, fluorescence microscopy under the microscope not
With developmental stage fluorescent protein expression situation.
Embodiment 3:
A kind of labeling method of Sepiella maindroni archaeocyte, uses turbot Vasa gene for marker gene,
Including,
1) the Sepiella maindroni fertilized eggs of 1- cell stage are collected, the specific steps are the same as those in embodiment 1；
2) 3 ' the UTR fragment products of amplification Vasa of double digestion insertion PGEM-T easy carrier and/or EGFP gene amplification
Product, connection, the specific steps are the same as those in embodiment 1；
3) products therefrom Sal I enzyme linearization process will be connected, and synthesizes 3 ' UTR mRNA of Vasa in vitro, wherein body
Outer synthesis is carried out using the external synthetic agent box of RNA；The step of external synthesis of 3 ' UTR mRNA of Vasa are as follows: 35 DEG C,
After cultivating 1.5h during 80rpm magnetic agitation, the MgCl of 80 μ L 1M is added2With the RNA Ⅲ of 15 μ L 0.3mM, hold
Continuous reaction 3.5h；
4) 3 ' UTR mRNA of gained Vasa DEPC water, 0.02% phenol red solution are diluted to 100ng/ μ l, be injected in by
Smart ovum is marked, at the same mark after fertilized eggs in embryo medium in 27 DEG C cultivate, fluorescence microscopy under the microscope not
With developmental stage fluorescent protein expression situation.
Comparative example 1:
A kind of labeling method of Sepiella maindroni archaeocyte, uses turbot Vasa gene for marker gene,
Including,
1) the Sepiella maindroni fertilized eggs of 1- cell stage are collected, the specific steps are the same as those in embodiment 1；
2) 3 ' the UTR fragment products of amplification Vasa of double digestion insertion PGEM-T easy carrier and/or EGFP gene amplification
Product, connection, the specific steps are the same as those in embodiment 1；
3) products therefrom Sal I enzyme linearization process will be connected, and synthesizes 3 ' UTR mRNA of Vasa in vitro, wherein body
Outer synthesis is carried out using the external synthetic agent box of RNA；The step of external synthesis of 3 ' UTR mRNA of Vasa are as follows: 38 DEG C,
6.0h is cultivated under 100rpm magnetic agitation；
4) 3 ' UTR mRNA of gained Vasa DEPC water, 0.02% phenol red solution are diluted to 100ng/ μ l, be injected in by
Smart ovum is marked, at the same mark after fertilized eggs in embryo medium in 27 DEG C cultivate, fluorescence microscopy under the microscope not
With developmental stage fluorescent protein expression situation.
Comparative example 2:
A kind of labeling method of Sepiella maindroni archaeocyte, uses turbot Vasa gene for marker gene,
Including,
1) the Sepiella maindroni fertilized eggs of 1- cell stage are collected, the specific steps are the same as those in embodiment 1；
2) 3 ' the UTR fragment products of amplification Vasa of double digestion insertion PGEM-T easy carrier and/or EGFP gene amplification
Product, connection, the specific steps are the same as those in embodiment 1；
3) products therefrom Sal I enzyme linearization process will be connected, and synthesizes 3 ' UTR mRNA of Vasa in vitro, wherein body
Outer synthesis is carried out using the external synthetic agent box of RNA；The step of external synthesis of 3 ' UTR mRNA of Vasa are as follows: 38 DEG C,
After cultivating 2.0h during 100rpm magnetic agitation, the MgCl of 90 μ L 1M is added2, sustained response 4.0h；
4) 3 ' UTR mRNA of gained Vasa DEPC water, 0.02% phenol red solution are diluted to 100ng/ μ l, be injected in by
Smart ovum is marked, at the same mark after fertilized eggs in embryo medium in 27 DEG C cultivate, fluorescence microscopy under the microscope not
With developmental stage fluorescent protein expression situation.
Comparative example 3:
A kind of labeling method of Sepiella maindroni archaeocyte, uses turbot Vasa gene for marker gene,
Including,
1) the Sepiella maindroni fertilized eggs of 1- cell stage are collected, the specific steps are the same as those in embodiment 1；
2) 3 ' the UTR fragment products of amplification Vasa of double digestion insertion PGEM-T easy carrier and/or EGFP gene amplification
Product, connection, the specific steps are the same as those in embodiment 1；
3) products therefrom Sal I enzyme linearization process will be connected, and synthesizes 3 ' UTR mRNA of Vasa in vitro, wherein body
Outer synthesis is carried out using the external synthetic agent box of RNA；The step of external synthesis of 3 ' UTR mRNA of Vasa are as follows: 38 DEG C,
After cultivating 2.0h during 100rpm magnetic agitation, the RNA Ⅲ of 20 μ L 0.3mM, sustained response 4.0h are added；
4) 3 ' UTR mRNA of gained Vasa DEPC water, 0.02% phenol red solution are diluted to 100ng/ μ l, be injected in by
Smart ovum is marked, at the same mark after fertilized eggs in embryo medium in 27 DEG C cultivate, fluorescence microscopy under the microscope not
With developmental stage fluorescent protein expression situation.
Embodiment 4:
Measure purpose RNA amount obtained by the external synthesis of 3 ' UTR mRNA of Vasa in embodiment 1,2,3 and comparative example 1,2,3
Account for the percentage of transcription gained total serum IgE amount.
The polyacrylamide gel (urea containing 7M/L) of preparation 12% uses the small-sized Vertial electrophorestic tank of Bio-rad, setting
For power 20W, electrophoresis time 15 minutes；After being transferred to culture dish EB dyeing after running glue, 15 minutes are stood, is used
Bio-rad observation of use instrument result simultaneously shoots photo, estimates under same background to the concentration of gained band, then acquires each band
Concentration relative percentages obtain the relative value of each band that is, by the photodensitometry to detection figure, then calculate transcription
Gained purpose RNA amount accounts for the percentage of transcription gained total serum IgE amount, draws embodiment 1,2,3 and the corresponding purpose of comparative example 1,2,3
The change curve of RNA percentage, as shown in Figure 1.1,2,3,4,5,6 respectively represent embodiment 1, embodiment 2, embodiment in Fig. 1
3, comparative example 1, comparative example 2, comparative example 3, as can be seen from the figure: embodiment 1, embodiment 2 and embodiment 3 synthesize Vasa in vitro
The percentage that purpose RNA amount accounts for transcription gained total serum IgE amount in 3 ' UTR mRNA is higher than comparative example 1, comparative example 2, comparative example 3,
This illustrates MgCl2It is needed with RNA Ⅲ and magnetic agitation condition combines, it is higher that transcription amount height, purity could be obtained
Purpose Vasa3 ' UTR mRNA, to improve the accuracy of labeling method of the present invention.
Embodiment 5:
A kind of labeling method of Sepiella maindroni archaeocyte, uses carp Vasa gene for marker gene, packet
It includes,
1) the Sepiella maindroni fertilized eggs of 1- cell stage are collected, the specific steps are the same as those in embodiment 1；
2) 3 ' the UTR fragment products of amplification Vasa of double digestion insertion PGEM-T easy carrier and/or EGFP gene amplification
Product, connection, the specific steps are the same as those in embodiment 1；
3) products therefrom Sal I enzyme linearization process will be connected, and synthesizes 3 ' UTR mRNA of Vasa in vitro, wherein body
Outer synthesis is carried out using the external synthetic agent box of RNA；The step of external synthesis of 3 ' UTR mRNA of Vasa are as follows: 38 DEG C,
After cultivating 2.0h during 100rpm magnetic agitation, the MgCl of 90 μ L 1M is added2With the RNA Ⅲ of 20 μ L 0.3mM,
Sustained response 4.0h；
4) 3 ' UTR mRNA of gained Vasa DEPC water, 0.02% phenol red solution are diluted to 100ng/ μ l, be injected in by
Smart ovum is marked, at the same mark after fertilized eggs in embryo medium in 27 DEG C cultivate, fluorescence microscopy under the microscope not
With developmental stage fluorescent protein expression situation.
Embodiment 6:
A kind of labeling method of Sepiella maindroni archaeocyte, uses Tilapia mossambica Vasa gene for marker gene,
Including,
1) the Sepiella maindroni fertilized eggs of 1- cell stage are collected, the specific steps are the same as those in embodiment 1；
2) 3 ' the UTR fragment products of amplification Vasa of double digestion insertion PGEM-T easy carrier and/or EGFP gene amplification
Product, connection, the specific steps are the same as those in embodiment 1；
3) products therefrom Sal I enzyme linearization process will be connected, and synthesizes 3 ' UTR mRNA of Vasa in vitro, wherein body
Outer synthesis is carried out using the external synthetic agent box of RNA；The step of external synthesis of 3 ' UTR mRNA of Vasa are as follows: 38 DEG C,
After cultivating 2.0h during 100rpm magnetic agitation, the MgCl of 90 μ L 1M is added2With the RNA Ⅲ of 20 μ L 0.3mM,
Sustained response 4.0h；
4) 3 ' UTR mRNA of gained Vasa DEPC water, 0.02% phenol red solution are diluted to 100ng/ μ l, be injected in by
Smart ovum is marked, at the same mark after fertilized eggs in embryo medium in 27 DEG C cultivate, fluorescence microscopy under the microscope not
With developmental stage fluorescent protein expression situation.
The prior art of routine techniques dawn known to those skilled in the art in above-described embodiment, therefore herein no longer in detail
It repeats.
The above embodiments are only used to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this field
Member can also make a variety of changes and modification without departing from the spirit and scope of the present invention.Therefore, all equivalent
Technical solution also belong to scope of the invention, scope of patent protection of the invention should be defined by the claims.
Sequence table
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<120>a kind of labeling method of Sepiella maindroni archaeocyte
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<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atcagctatt taattatcgg agatc 25
<210> 3
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tagctgacgt cgccccatgg ttagc 25
<210> 4
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tatgtcgcgg ttacttgcac agtcgtc 27
Claims (10)
1. a kind of labeling method of Sepiella maindroni archaeocyte, uses Vasa gene for marker gene, feature exists
In: including,
Collect the Sepiella maindroni fertilized eggs of 1- cell stage；
Double digestion is inserted into amplification Vasa3 ' the UTR fragment products and/or EGFP gene amplified production of PGEM-T easy carrier, even
It connects；
Products therefrom Sal I enzyme linearization process will be connected, and synthesizes Vasa3 ' UTR mRNA in vitro；
Gained Vasa3 ' UTR mRNA is injected in fertilized eggs to be marked.
2. a kind of labeling method of Sepiella maindroni archaeocyte according to claim 1, it is characterised in that: institute
It states Vasa gene and is selected from zebra fish Vasa gene, Tilapia mossambica Vasa gene, turbot Vasa gene, carp Vasa gene or green Medaka
Vasa gene.
3. a kind of labeling method of Sepiella maindroni archaeocyte according to claim 1, it is characterised in that: institute
State Vasa3 ' UTR fragment amplification primer are as follows:
Vasa-F:5 '-GCTGCTGATGTCTAATGCGATT-3 '；
Vasa-R:5 '-ATCAGCTATTTAATTATCGGAGATC-3 '.
4. a kind of labeling method of Sepiella maindroni archaeocyte according to claim 1, it is characterised in that: institute
State Vasa3 ' UTR fragment amplification step are as follows:
Genome total serum IgE is extracted, reverse transcription synthesizes cDNA, carries out PCR amplification；
The response procedures of the PCR amplification are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 70s, 30 circulations；
Last 72 DEG C of extensions 10min.
5. a kind of labeling method of Sepiella maindroni archaeocyte according to claim 4, it is characterised in that: institute
It states Total RNAs extraction and hydroxychloroquine sulfate is added after tissue sample grinding in the process to 1-2 μM, then in frequency 2-4kHz, intensity
12-15mW/cm2Ultrasonic wave under carry out ultrasonic wave circulation stimulation after be centrifuged again.
6. a kind of labeling method of Sepiella maindroni archaeocyte according to claim 1, it is characterised in that: institute
State EGFP gene amplification primers are as follows:
EGFP-F:5 '-TAGCTGACGTCGCCCCATGGTTAGC-3 '；
EGFP-R:5 '-TATGTCGCGGTTACTTGCACAGTCGTC-3 '.
7. a kind of labeling method of Sepiella maindroni archaeocyte according to claim 1, it is characterised in that: institute
State introducing Aat II and Sac II restriction enzyme site in EGFP gene amplified production.
8. a kind of labeling method of Sepiella maindroni archaeocyte according to claim 1, it is characterised in that: institute
It states and is diluted to 100ng/ μ l with DEPC water, 0.02% phenol red solution before 3 ' UTR mRNA are injected.
9. a kind of labeling method of Sepiella maindroni archaeocyte according to claim 1, it is characterised in that: institute
The step of stating the external synthesis of Vasa3 ' UTR mRNA are as follows: cultivate 1.5- during 35-40 DEG C, 80-130rpm magnetic agitation
After 2.5h, the MgCl of 80-100 μ L1M is added2With the RNA Ⅲ of 15-22 μ L 0.3mM, sustained response 3.5-4.5h.
10. a kind of labeling method of Sepiella maindroni archaeocyte according to claim 1, it is characterised in that:
Fertilized eggs after the label are cultivated in embryo medium in 25-30 DEG C, in fluorescence microscopy microscopic observation different development stage
Fluorescent protein expression situation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724744A (en) * | 2019-09-16 | 2020-01-24 | 浙江海洋大学 | Marking method of male sepiella maindroni germ line cells |
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2019
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724744A (en) * | 2019-09-16 | 2020-01-24 | 浙江海洋大学 | Marking method of male sepiella maindroni germ line cells |
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