CN104611369A - Method for marking nerve cell through electroporation auxiliary gene transfer - Google Patents
Method for marking nerve cell through electroporation auxiliary gene transfer Download PDFInfo
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Abstract
The invention discloses a method for marking nerve cells through electroporation auxiliary gene transfer. The method comprises the following steps: designing and manufacturing an electrode of an electrode device, and establishing and preparing plasma containing a target gene or a RNA interface fragment; designing an electrophotoluminescence pulse condition; injecting plasmid into a third ventricle, namely, injecting the plasma into the third ventricle of a rat or a mouse by using a microscopic injector; transferring the plasma to mark nerve cells in appointed positions, connecting the electrode with a point stimulation generator, and under the action of an electric field which is positive inside and negative outside, transfecting the plasmid into nerve cells of the anterior subventricular zone of the rat or the mouse. By adopting the method, the genetic transfection inside brains of living animals can be directionally controlled, and the defects such as cell immunogenicity, potential pathogenicity and tumorigenicity caused by virus transgenosis are also avoided. The method has the advantages of being simple and convenient to operate, efficient, traceable and the like, transfection of multiple types of plasmid can be simultaneously achieved, and the defects of a conventional method can be overcome.
Description
Technical field
The invention belongs to neurobiology technical field, be specifically related to one and utilize the neuronic method of electroporation auxiliary gene metastatic marker.
Background technology
In neural system fetal development, neuronal migration is an extremely important phenomenon, abnormal (the Neuronal migration disorders of neuronal migration, NMD) various cortex heteroplasia disease will be caused, as congenital agyria, epilepsy, amentia etc., it is the main pathological basis of insoluble multiple sacred disease in physianthropy.
The middle serotonergic neuron at Mammals olfactory bulb place is that embryo starts to occur on the 14th day the earliest, but olfactory bulb can constantly receive new neurone in life.The neural hyperplasia of this adult is conservative in evolution, all exists from Reptilia to Mammals.The more new phenomenon of the middle serotonergic neuron of this local is presented district in the central authorities of insect and Crustacean and is also existed.
In Mammals, in the middle of new olfactory bulb, serotonergic neuron is from forebrain sub-ependymal layer (subventricular zone, SVZ) district, room origin.SVZ is positioned at the wall side of third ventricle, is a specific region of forebrain after Mammals birth.This specific neuronal precursor, is also called SVZa cell, is positioned at the SVZ district on front side of third ventricle.SVZa cell can generate new neurone, also referred to as neuroblast or type A cell as precursor cell.This neuroblast moves to olfactory bulb with the catenulate pattern of rows and columns of tangent migration pattern shape, this migration channel along head end is called head end migration channel (rostral migratory stream, RMS), extend the large-scale path as olfactory bulb central authorities always, reach several millimeter, and the diameter of neuroblast of migration own only have 10 microns.Grow into serotonergic neuron in the middle of ripe inhibition when these neuroblasts move to after in olfactory bulb, be integrated in the existing neural network of olfactory bulb and play function.Therefore neuroblast is studied extremely important for the integration of illustrating neural circuitry by the transition process of RMS approach.Because SVZ district is positioned at the depths of brain, which has limited the mark to this district's cell or operation.Therefore existing research method is all observe after putting to death animal section, can not accurately locate and follow the trail of the specific neuroblast of this group, and can not carry out efficient gene operation.
Along with the fast development of medical science, biological Science and Technology, increasing evidence shows that normal growth course and numerous disease are all relevant with the structure of gene or function, thus studies from gene level growth course and carry out gene therapy further to have great importance.Simultaneously for research growth course or gene therapy, all need therefore to be necessary very much to research and develop a kind of directional technology that can carry out marking and carrying out genetic manipulation in living animal, existence and the later stage that can not affect animal grow simultaneously.Electroporation transgenic method is the transgenic method of a kind of physical property, non-viral, principle is that specific pulsed electrical field is instantaneous reversibly changes membrane passage by utilizing, form the perforation channels of diameter 20 ~ 100nm scope on film, make genetic stew proceed to cell.From the successful dependence of electroporation auxiliary gene rotaring dyeing technology at live body mammal skeletal muscle of reporting for work, the method finds application successively in the biological tissues such as skin, liver kidney, retina.The method is easy to operate and safe, transfection efficiency is high, the advantages such as targeting is strong, but due to the electrical characteristic difference of different tissues cell, the percentage of perforation of cytolemma and degree of perforation have the dependency of Field signature parameter, therefore need appropriate design electrode, determine the electric pulsing conditions be applicable to, and location and directivity electric field give exactly, thus realize the effective gene transfection to object cell, reduce simultaneously or avoid the damage to its hetero-organization of surrounding.Therefore concerning integumentary musculature retina etc. is in the tissue on animal top layer, relatively simple, and SVZ district is in the depths of brain, and SVZa district is a specific circumscribed position, therefore need to design the direction that the electrode and electric pulse parameter that meet age bracket rat head size and curvature and electric field give.
Summary of the invention
The object of the invention is the defect overcoming prior art, one is provided to utilize the neuronic method of electroporation auxiliary gene metastatic marker, the inventive method is easy and simple to handle, efficient, to advantages such as animal injury are little, and the transfection of multiple plasmid can be realized, as gene overexpression plasmid, RNA interference plasmid etc. simultaneously.
The technical solution adopted in the present invention is, one utilizes the neuronic method of electroporation auxiliary gene metastatic marker, specifically implements according to following steps:
The electrode of step 1, designing and making electrode device: according to the electrode of the large little makings different size of the head of required rat and mouse;
Step 2, structure and preparation contain the plasmid of goal gene or RNA interference fragment;
Step 3, design electric stimulation pulse condition;
Step 4, will to build and the plasmid prepared is injected into third ventricle, the plasmid using microinjector step 2 to be built and prepare is injected in the third ventricle of rat or mouse;
Step 5, plasmid metastatic marker specified location neurone, by electrode connection points stimulus generator, under interior just outer negative electric field action, plasmid transfection is entered in the neurocyte of rat or mouse forebrain sub-room ependymal layer, and with the electric stimulation pulse conditioned stimulus rat designed in step 3 or mouse SVZa district neuronal cell layers.
Feature of the present invention is also,
The two poles of the earth of electrode device are connected with electric stimulus generator by plug, shape, the size of electrode conform to corresponding age, laboratory animal head size, curvature, laboratory animal selects E16 and 16 days embryonic stages respectively, namely P0 is born latter 0 day, the animal that is namely born latter 10 days of P10, concrete specification is as follows, wherein, electrode radius mm:
The plasmid containing goal gene or RNA interference fragment of step 2 is linked by cDNA and the pSUPER vector plasmid of goal gene or RNA interference fragment.
Microinjector is prepared in the following manner: draw instrument with PN-30 electrode, by diameter 1.0mm under the condition of temperature 70 C, the glass capillary of long 75mm pulls into needle-like, is connected by pin, prepares microinjector with medical rubber pipe.
Electric stimulation pulse condition in step 3 is voltage 60V, pulsewidth 50ms, recurrent interval interval 100ms, and 5 pulses are one group.
Plasmid metastatic marker specified location neurone, by electrode connection points stimulus generator, under interior just outer negative electric field action, plasmid transfection is entered in the neurocyte of rat or mouse forebrain sub-room ependymal layer and be specially: by electrode connection points stimulus generator, its negative pole is in the head end of laboratory animal, positive pole is in the pillow rear of laboratory animal, cathode and anode directions is relative, under the stimulation conditions of step 3, by under electronegative plasmid outside negative interior positive electric field action, be transfected into SVZa district neuronal cell layer.
The invention has the beneficial effects as follows:
(1) determine the electrode placement positions that is applicable to and angle: because SVZa district is positioned at the deep regions of brain, when therefore determining that electricity turns the position of electrode and angle extremely important.The negative pole of electrode is in the head end of laboratory animal by the present invention, and positive pole is in the pillow rear of laboratory animal, and cathode and anode directions is relative, can will be injected into the electronegative plasmid of the ventricles of the brain effectively and accurately, shift to SVZa district cell.
(2) optimal electric pulse parameter is determined: electroporation is a kind of physical property transgenic method, and due to the electrical characteristic difference of different tissues cell, the percentage of perforation of cytolemma and degree of perforation all depend on Field signature parameter.Electroporation also can cause damage to cell simultaneously, therefore needs Field signature parameter reasonable in design.The present invention, by many group parameter detecting and trial, determines the electric pulse parameter of the most applicable SVZa district cell, i.e. 60V, 50ms, 5 times, interval 100ms.
(3) present method can carry out genetic manipulation to brephic laboratory animal, and can observe constantly after life, is conducive to the scientific research of auxology aspect.
The inventive method establishes one in living animal brain, utilizes the neuronic method of electroporation auxiliary gene metastatic marker, transfection in the directive controlling gene brain of energy, genetic marker operation can be carried out to embryonic stage, postnatal laboratory animal, both avoided the drawback such as cellular immunization source property that viral transgene brings and potential pathogenic, tumorigenicity, and can only mark specific cell colony again and not affect the cell of surrounding.The inventive method has the advantages such as easy and simple to handle, efficient, can need to design different schemes according to different research, and can realize the transfection of multiple plasmid, achieves the transgenosis in brain district, deep.The present invention is neuroscience, grow the channel genes of scientific research and cell tracker provides novel method, and provides new approaches for the gene therapy of encephalopathy.
Accompanying drawing explanation
Fig. 1 is electrode placement positions of the present invention and angle schematic diagram, and wherein, A, B are electrode placement positions and angle, laboratory animal head is placed between electrode, the negative pole of electrode is in the head end of laboratory animal, and positive pole is in the pillow rear of laboratory animal, and cathode and anode directions is relative;
Fig. 2 is that SVZa cell is at the schematic diagram moved to olfactory bulb; SVZa: the sub-room ependymal layer anterior subventricular zone of forebrain; RMS: rostral migration stream Rostra Migratory Stream; WM: white matter white matter; SEZ: subependymal region subependymal zone; Gcl: GCL granule cell layer; Mcl: MCL mitral cell layer; Epl: outer plexiform layer externalplexiform layer; Gl: olfactory glomerulus layer glomerular layer; Onl: olfactory nerve layer olfactory nervelayer;
Fig. 3 is the neuronic distribution situation of P2 time point after brain sagittal slices observation Scramble siRNA-EGFP transfection;
Fig. 4 is the neuronic distribution situation of P8 time point after brain sagittal slices observation Scramble siRNA-EGFP transfection;
Fig. 5 is neuronic distribution situation statistics after brain sagittal slices observes Scramble siRNA-EGFP transfection, wherein, and SVZa: the sub-room ependymal layer anterior subventricular zone of forebrain; RMS: rostral migration stream Rostra Migratory Stream; SEZ: subependymal region subependymal zone; GCL: GCL granule cell layer; MCL: MCL mitral cell layer; EPL: outer plexiform layer external plexiform layer; GL: olfactory glomerulus layer glomerular layer;
Fig. 6 is the neuronic distribution situation of P14 time point after brain sagittal slices observation EGFP transfection;
Fig. 7 is the neuronic form of P14 time point after laser co-focusing observation EGFP transfection;
Fig. 8 is the neuronic distribution situation of P14 time point after olfactory bulb coronal section observation EGFP transfection;
After Fig. 9 olfactory bulb coronal section observes EGFP transfection, the neuronic distribution situation of P14 is added up, wherein, and SEZ: subependymal region subependymal zone; GCL: GCL granule cell layer; MCL: MCL mitral cell layer; GL: olfactory glomerulus layer glomerular layer.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.
The invention provides one and utilize the neuronic method of electroporation auxiliary gene metastatic marker, particularly relate to a kind of utilize the perforation auxiliary gene transfer techniques mark neuronic electrode special device in brain SVZa district and pulse parameter and electrode user to.
The present invention adopts special electrode device, determine optimum electric stimulation pulse condition, utilize the method for electroporation by the birth of the plasmid transfection containing green fluorescent protein (enhanced green fluorescence protein, EGFP) the rat SVZa district cell of latter 0 ~ 2 day.
The inventive method comprises the steps:
1, designing and making electrode
In the present invention, described laboratory animal is selected from mouse, rat.
In the present invention, described laboratory animal selects 16 days embryonic stages (E16) respectively, be born latter 0 day (P0), be born latter 10 days (P10).The laboratory animal at other ages selects electrode and working method according to close age bracket.
In the present invention, according to head size and the curved transition of often kind of animal different ages, for often kind of animal designs and produces electrode, concrete specification following (unit: electrode radius mm):
2, build and prepare the plasmid containing goal gene or RNA interference fragment, this plasmid can design and carry goal gene as fluorescin so that spike.
Plasmid construction containing goal gene: according to goal gene sequent synthesis gene fragment, design and synthesize primer, PCR is utilized to reflect amplifying target genes, electrophoresis also reclaims the cDNA obtaining goal gene, by goal gene and vector plasmid, as pcDNA3.1 carries out connecting, transforming, and PCR screens bacterium colony, then row plasmid extraction is dropped into positive bacteria, qualification, order-checking; The plasmid of structure is diluted to 3mg/ml concentration for subsequent use.
Plasmid construction containing RNA interference fragment: according to siRNA standard, siRNA sequence is designed and synthesized for goal gene coding region, the annealing of strand goal gene fragment is connected to form double-stranded DNA, with vector plasmid, as pSUPER carries out connecting, transforming, and PCR screens bacterium colony, then drops into row plasmid extraction to positive bacteria, qualification, order-checking; The plasmid of structure is diluted to 3mg/ml concentration for subsequent use.
3, electric stimulation pulse condition is designed
In the scope of safe electric field, design voltage scope is from 20V to 100V, 25 groups of electric pulse stimulation conditions of pulsewidth 20ms to 100ms, detected by brain sagittal slices, immunohistochemical staining or Western blot, determine with voltage 60V, pulsewidth 50ms, recurrent interval interval 100ms, 5 pulses are that the Parameter Conditions of a group is as SVZa district neuronal cell layers optimum electric pulse stimulation condition.
4, experimentation on animals
Experimentation on animals embryonic stage, for E16 rat: the pregnant mouse of SD of getting conceived E16 days, with 10% Chloral Hydrate (by body weight 350mg/Kg dosage) abdominal injection, anesthesia, after pregnant mouse anesthesia completely, its belly is upwards placed on flat board, carries out surface sterilization with alcohol swab; Cover rat with aseptic gauze, in the middle of gauze, open the openning of 3-5 cm, opening is aimed at ventrimeson side; With dissecting scissors in rat abdomen distance ventrimeson about 0.5 centimeters, the openning of opening 2.5 cm parallel with ventrimeson; Then open abdominal cavity, expose uterus, uterus is pulled out from abdominal cavity, be placed on gauze.Control mouse embryo with finger-like tweezer, plasmid is entered by glass-micropipe microinjection in the third ventricle of tire mouse, injection 2-3 μ l.Be placed between electrode by tire mouse head, with 60V, 50ms, 5 times, the voltage of interval 100ms carries out electricity and turns.By Zhi Hui abdominal cavity, uterus after electricity turns, continued to raise by female mouse after stitching, ripe to about E21 days to fetal development, namely female mouse can give birth to tire mouse.
Postnatal animal is tested, and for P0 rat: the SD rat of getting P0 days, anaesthetizes on ice.After anesthesia completely, just plasmid is entered by glass-micropipe microinjection in the third ventricle of tire mouse, injection 2-3 μ l.Be placed between electrode by tire mouse head, with 60V, 50ms, 5 times, the current field condition of interval 100ms carries out electricity and turns.
5, electroporation auxiliary gene transfection
By the Electrode connection point stimulation electric stimulus generator (BTX T830 model electroporation) be applicable to, its negative pole is in the head end of laboratory animal, positive pole is in the pillow rear of laboratory animal, cathode and anode directions relatively (Fig. 1), under above-mentioned stimulation conditions, by under electronegative plasmid outside negative interior positive electric field action, be transfected into SVZa district neuronal cell layer.
The present invention is with at body electroporation auxiliary gene transfection NMDA RNAi-EGFP gene E16, P0 rat SVZa neuronal cell layers as an example, obtain best electric pulse stimulation condition, for 60V, 50ms, 5 times, interval 100ms, and successfully follow the trail of this group of specific neurocyte and the function of goal gene can be used for, there is the feature of efficient, low side effect, economical and convenient.
The present invention's electrode device used designs electrode parameter according to the laboratory animal of different sorts and different ages, to meet size and the curvature of head.Like this can handled easily, ensure to cover with the note of brain also to reduce animal injury.
Embodiment 1
The present embodiment assists Scramble siRNA-EGFP gene transfection E16 embryonic stage rat SVZa district cell with electroporation, observes early stage neurone RMS transition process, as an example, elaborates the present invention.
1, plasmid vector is built
Design scramble siRNA, the sequence of design 19 Nucleotide, concrete sequence is 5 '-CAGTCGCGTTTGCGACTGG-3 '.The annealing of strand goal gene fragment is connected to form double-stranded DNA.By the linearizing of pSUPER plasmid vector: BglII and HindIII double digestion pSUPER plasmid vector, makes its linearizing, 1% sepharose reclaims large fragment.By linearizing pSUPER plasmid vector with annealing fragment in the effect of T4 ligase enzyme, 4 DEG C of connections are spent the night, and it is heat-shock transformed to DH5 α competence bacterium to connect product 42 DEG C, and that microbiotic of card-coating is dull and stereotyped, 37 DEG C of overnight incubation.Select mono-clonal bacterium colony, in a small amount extracting plasmid, enzyme cut, qualification of checking order formal after, with the EndoFree Plasmid Maxi a large amount of extracting of Kit test kit and plasmid purification, detect OD260/280 value, after qualified, plasmid be diluted to 3mg/ml concentration for subsequent use.
2, microinjector preparation
Draw instrument with PN-30 electrode, by diameter 1.0mm under the condition of temperature 70 C, the glass capillary of long 75mm pulls into needle-like.Pin is connected with medical rubber pipe.
3, the injection of laboratory animal brain plasmid and electroporation transfection
Get the pregnant mouse of SD of conceived E16 days, with 10% Chloral Hydrate (by body weight 350mg/Kg dosage) abdominal injection, anesthesia.After pregnant mouse anesthesia completely, its belly is upwards placed on flat board, carries out surface sterilization with alcohol swab; Cover rat with aseptic gauze, in the middle of gauze, open the openning of 3-5 cm, opening is aimed at ventrimeson side; With dissecting scissors in rat abdomen distance ventrimeson about 0.5 centimeters, the openning of opening 2.5 cm parallel with ventrimeson; Then open abdominal cavity, expose uterus, uterus is pulled out from abdominal cavity, be placed on gauze.Control mouse embryo with finger-like tweezer, plasmid is entered by glass-micropipe microinjection in the third ventricle of tire mouse, injection 2-3 μ l.Be placed between electrode by tire mouse head, use the large small electrode of 3.0mm in this example, the negative pole of electrode is in the head end of laboratory animal, and positive pole is in the pillow rear of laboratory animal, and cathode and anode directions is relative, and with 60V, 50ms, 5 times, the voltage of interval 100ms carries out electricity and turns.By Zhi Hui abdominal cavity, uterus after electricity turns, continued to raise by female mouse after stitching, ripe to about E21 days to fetal development, namely female mouse can give birth to tire mouse.
4, pour into, draw materials and film-making
After surgery, take out the newborn rat of raw rear each time point respectively, on ice after anesthesia, after heart perfusion, get brain with 4% paraformaldehyde (PFA)-0.1mol/L phosphate buffered saline buffer (pH7.4) of physiological saline and 4 DEG C rapidly, 30% sucrose dehydration is until tissue cuts into slices (20-25mm) in-20 DEG C of capable sagittal sections of cryostat freezing microtome after sinking to the bottom.Sagittal slices need comprise olfactory bulb and third ventricle.
5, Fluorescent immunohistochemistry
Tissue slice is after 0.2%Triton X-100 punches and normal sheep serum is closed, add an anti-rabbit anti-green fluorescent protein (GFP) 1:500 (Molecular probe, A11122) latter 4 DEG C are spent the night, after use two anti-Alexa488 (Molecular probe) 1:1000 and DAPI (4 ', 6 '-diamidino-2-phenylindole, Sigma, D9542) transfect cell core, row Fluorescent immunohistochemistry detects, and uses Zeiss confocal microscopy after mounting.
Result display (see Fig. 1-5), under adopting the electrode of the inventive method and the effect of Field signature parameter, can by smooth for scramble siRNA-EGFP plasmid energy transfection on the brain SVZa district neuronal cell of SD pregnant mouse embryo mouse, by sagittal section section, dyeing carry out follow-up observation and research, the inventive method is prior art comparatively, has the advantages such as accurate positioning, fluorescent mark, genetic manipulation, repeatable strong, economical and convenient.
As shown in Figure 1, A, B electrode placement positions and angle, be placed in laboratory animal head between electrode, and the negative pole of electrode is in the head end of laboratory animal, and positive pole is in the pillow rear of laboratory animal, and cathode and anode directions is relative.Fig. 2 is the schematic diagram SVZa that SVZa cell is moving to olfactory bulb: the sub-room ependymal layer anterior subventricular zone of forebrain; RMS: rostral migration stream Rostra Migratory Stream; WM: white matter white matter; SEZ: subependymal region subependymal zone; Gcl: GCL granule cell layer; Mcl: MCL mitral cell layer; Epl: outer plexiform layer externalplexiform layer; Gl: olfactory glomerulus layer glomerular layer; Onl: olfactory nerve layer olfactory nervelayer.
As shown in Figure 3, after electroporation transfection plasmid is carried out to E16 tire mouse, sagittal slices observation is carried out at P2 days, take black as base plate (same with figure below), the neurone that green fluorescent label (white portion namely in Fig. 3) is EGFP plasmid in transfection, blue (grey parts namely in Fig. 3) is DAPI phaeochrome cell core.The SVZa district neurocyte can seen on E16 days marks moves along RMS approach to olfactory bulb.
As shown in Figure 4, after electroporation transfection plasmid is carried out to E16 tire mouse, carried out sagittal slices observation at P8 days, the neurone that green fluorescent label (white portion namely in Fig. 4) is EGFP plasmid in transfection, blue (grey parts namely in Fig. 4) is DAPI phaeochrome cell core.Visible when P8 days, most nerve cell migration also enters olfactory bulb, is integrated into the at all levels of olfactory bulb.
As shown in Figure 5, after electroporation transfection plasmid is carried out to E16 tire mouse, add up respectively neurone on green fluorescence (white portion) albumen EGFP mark at all levels in brain in distribution situation.When can see P2, most EGFP positive cell is positioned at SVZ, RMS district, and small part arrives the SEZ district of olfactory bulb central authorities, and during to P8, most EGFP positive cell enters olfactory bulb and is incorporated into the at all levels of olfactory bulb, as GCL, MCL, EPL etc.
Embodiment 2
BALB/c mouse P2 phase SVZa district cell after the present embodiment assists EGFP gene transfection to be born with electroporation, observes the rear neurone RMS migration results of birth, as an example, elaborates the present invention.
1, plasmid vector is built
With the EndoFree Plasmid Maxi a large amount of extracting of Kit test kit and purifying EGFP plasmid, detect OD260/280 value, after qualified, plasmid is diluted to 3mg/ml concentration for subsequent use.
2, microinjector preparation
Draw instrument with PN-30 electrode, by diameter 1.0mm under the condition of temperature 70 C, the glass capillary of long 75mm pulls into needle-like.Pin is connected with medical rubber pipe.
3, the injection of laboratory animal brain plasmid and electroporation transfection
Get the BALB/c mouse of P2 days, with 10% Chloral Hydrate (by body weight 350mg/Kg dosage) abdominal injection, anesthesia.After anesthesia completely, carry out brain surface sterilization with alcohol swab, control mouse with finger-like tweezer, plasmid is entered in the third ventricle of mouse by glass-micropipe microinjection, injection 2-3 μ l.Be placed between electrode by tire mouse head, use the large small electrode of 1.75mm in this example, the negative pole of electrode is in the head end of laboratory animal, and positive pole is in the pillow rear of laboratory animal, and cathode and anode directions is relative, and with 60V, 50ms, 5 times, the voltage of interval 100ms carries out electricity and turns.After electricity turns, mouse is sent back to female mouse to continue to raise.
4, pour into, draw materials and film-making
After surgery, get the mouse of P14 days, on ice after anesthesia, after heart perfusion, brain is got rapidly with 4% paraformaldehyde (PFA)-0.1mol/L phosphate buffered saline buffer (pH7.4) of physiological saline and 4 DEG C, 30% sucrose dehydration until tissue sink to the bottom after in-20 DEG C of capable olfactory bulbs of cryostat freezing microtome continuous coronal section (20-25mm) and sagittal slices (20-25mm), sagittal slices need comprise olfactory bulb and third ventricle.
5, Fluorescent immunohistochemistry
Tissue slice is after 0.2%Triton X-100 punches and normal sheep serum is closed, add an anti-rabbit anti-green fluorescent protein (GFP) 1:500 (Molecular probe, A11122) latter 4 DEG C are spent the night, after use two anti-Alexa488 (Molecular probe) 1:1000 and DAPI (4 ', 6 '-diamidino-2-phenylindole, Sigma, D9542) transfect cell core, row Fluorescent immunohistochemistry detects, and uses Zeiss confocal microscopy after mounting.
Result display (see Fig. 6-9), under adopting the electrode of the inventive method and the effect of Field signature parameter, can by the brain SVZa district neuronal cell of BALB/c mouse after the smooth transfection of EGFP luciferase expression plasmid to birth, by the section of the coronal section of olfactory bulb, follow-up observation and research are carried out in dyeing, the inventive method is prior art comparatively, without the need to cutting mouse brain skin and skull, only adopt electrode injection and apply electric field, little to mouse wound, generally do not affect its development growth, have no side effect, there is accurate positioning simultaneously, fluorescent mark, genetic manipulation, repeatable strong, the advantages such as economical and convenient.
As shown in Figure 6, after electroporation transfection plasmid is carried out to the BALB/c mouse of P2, sagittal slices observation is carried out at P14 days, the neurone that green fluorescent label (white portion in figure) is EGFP plasmid in transfection, blue (grey parts in figure) is DAPI phaeochrome cell core.Visible when P14 days, most nerve cell migration also enters olfactory bulb, be integrated into the at all levels of olfactory bulb, and dendron germinates, is integrated in the neural circuitry of olfactory bulb.
As shown in Figure 7, amplified by Fig. 6 square frame place cell and observe, can see P14 days EGFP positive cells and stretch out apical dendrite to EPL layer, simultaneously dendron also germinates at the end, forms the form of serotonergic neuron in the middle of classical olfactory bulb.
As shown in Figure 8, after electroporation transfection plasmid is carried out to the BALB/c mouse of P2, the coronal section carrying out olfactory bulb at P14 days is observed, the neurone that green fluorescent label (white portion in figure) is EGFP plasmid in transfection, blue (grey parts in figure) is DAPI phaeochrome cell core.Visible when P14 days, most nerve cell migration also enters the at all levels of olfactory bulb, and dendron germinates, and is integrated in the neural circuitry of olfactory bulb.
As shown in Figure 9, after electroporation transfection plasmid is carried out to the BALB/c mouse of P2, the distribution situation during the neurone respectively in statistics green fluorescent protein EGFP mark (white portion in figure) is at all levels in olfactory bulb.When can see P14, most EGFP positive cell is positioned at GCL, GL layer, and all the other positive cells are distributed in MCL, EPL layer, and SEZ district also has a small amount of cell distribution.
Gene transfection in the directive control living animal brain of the present invention's energy, and realize in vivo marker tracking.The inventive method is easy and simple to handle, efficient, to advantages such as animal injury are little, and can realize the transfection of multiple plasmid, as gene overexpression plasmid, RNA interference plasmid etc. simultaneously.
The present invention adopts electrode device, determine the electric stimulation pulse condition be suitable for, utilize the method for electroporation by the plasmid transfection containing fluorescin in the neurocyte of sub-room ependymal layer (anterior subventricularzone, SVZa) of rat forebrain.The present invention can gene transfection in directive control living animal brain, avoids the drawback such as cellular immunization source property that viral transgene brings and potential pathogenic, tumorigenicity simultaneously.The inventive method has the advantages such as easy and simple to handle, efficient, and can realize the transfection of multiple plasmid simultaneously, as gene overexpression plasmid, RNA interference plasmid etc., has the advantages such as traceability simultaneously, compensate for the weak point of traditional method.The present invention is that the channel genes of Neuroscience Research provides can the method for reference, can be directly used in the correlative study of the gene function, proliferation and differentiation, cell migration etc. of brain SVZa district neurocyte, Study on Acceleration process for this area scientific research personnel.
Claims (6)
1. utilize the neuronic method of electroporation auxiliary gene metastatic marker, it is characterized in that, specifically implement according to following steps:
The electrode of step 1, designing and making electrode device: according to the electrode of the large little makings different size of the head of required rat and mouse;
Step 2, structure and preparation contain the plasmid of goal gene or RNA interference fragment;
Step 3, design electric stimulation pulse condition;
Step 4, will to build and the plasmid prepared is injected into third ventricle, the plasmid using microinjector step 2 to be built and prepare is injected in the third ventricle of rat or mouse;
Step 5, plasmid metastatic marker specified location neurone, by electrode connection points stimulus generator, under interior just outer negative electric field action, plasmid transfection is entered in the neurocyte of rat or mouse forebrain sub-room ependymal layer, and with the electric stimulation pulse conditioned stimulus rat designed in step 3 or mouse SVZa district neuronal cell layers.
2. according to claim 1ly utilize the neuronic method of electroporation auxiliary gene metastatic marker, it is characterized in that, the two poles of the earth of described electrode device are connected with electric stimulus generator by plug, shape, the size of electrode conform to corresponding age, laboratory animal head size, curvature, laboratory animal selects E16 and 16 days embryonic stages respectively, namely P0 is born latter 0 day, the animal that is namely born latter 10 days of P10, concrete specification is as follows, wherein, and electrode radius mm:
3. according to claim 1ly utilize the neuronic method of electroporation auxiliary gene metastatic marker, it is characterized in that, the plasmid containing goal gene or RNA interference fragment of described step 2 is linked by cDNA and the pSUPER vector plasmid of goal gene or RNA interference fragment.
4. according to claim 1ly utilize the neuronic method of electroporation auxiliary gene metastatic marker, it is characterized in that, described microinjector is prepared in the following manner: draw instrument with PN-30 electrode, by diameter 1.0mm under the condition of temperature 70 C, the glass capillary of long 75mm pulls into needle-like, pin is connected with medical rubber pipe, prepares microinjector.
5. according to claim 1ly utilize the neuronic method of electroporation auxiliary gene metastatic marker, it is characterized in that, the electric stimulation pulse condition in described step 3 is voltage 60V, pulsewidth 50ms, recurrent interval interval 100ms, and 5 pulses are one group.
6. according to claim 1ly utilize the neuronic method of electroporation auxiliary gene metastatic marker, it is characterized in that, described plasmid metastatic marker specified location neurone, by electrode connection points stimulus generator, under interior just outer negative electric field action, plasmid transfection is entered in the neurocyte of rat or mouse forebrain sub-room ependymal layer and be specially: by electrode connection points stimulus generator, its negative pole is in the head end of laboratory animal, positive pole is in the pillow rear of laboratory animal, cathode and anode directions is relative, under the stimulation conditions of step 3, by under electronegative plasmid outside negative interior positive electric field action, be transfected into SVZa district neuronal cell layer.
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| CN201510038228.4A Pending CN104611369A (en) | 2015-01-26 | 2015-01-26 | Method for marking nerve cell through electroporation auxiliary gene transfer |
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| CN108841865A (en) * | 2018-06-14 | 2018-11-20 | 新乡医学院 | Electric transgenic method, mouse spinal cord electricity turn histotomy culture model method for building up and its application in vitro for a kind of mouse spinal cord |
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| JP2002065112A (en) * | 2000-09-01 | 2002-03-05 | Japan Science & Technology Corp | Method for producing transgenic animal |
| KR20130079731A (en) * | 2012-01-03 | 2013-07-11 | 한림대학교 산학협력단 | Generation of human disease model including human periventricular nodular heterotopia by in utero electroporation of the lysophosphatidic acid receptor |
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| JP2002065112A (en) * | 2000-09-01 | 2002-03-05 | Japan Science & Technology Corp | Method for producing transgenic animal |
| KR20130079731A (en) * | 2012-01-03 | 2013-07-11 | 한림대학교 산학협력단 | Generation of human disease model including human periventricular nodular heterotopia by in utero electroporation of the lysophosphatidic acid receptor |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841865A (en) * | 2018-06-14 | 2018-11-20 | 新乡医学院 | Electric transgenic method, mouse spinal cord electricity turn histotomy culture model method for building up and its application in vitro for a kind of mouse spinal cord |
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