CN100443588C

CN100443588C - 'Jingning' chicken embryo fibroblast series and its culture method

Info

Publication number: CN100443588C
Application number: CNB2006101649838A
Authority: CN
Inventors: 关伟军; 马月辉; 刘长青; 包阿东; 王有柱; 刘学东
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2006-12-11
Filing date: 2006-12-11
Publication date: 2008-12-17
Anticipated expiration: 2026-12-11
Also published as: CN1995333A

Abstract

The invention discloses a high-living rate, high-purity chick static embryo phoroblast system with reserving number at CGMCC No. 1878, which is characterized by the following: culturing high-purity phoroblast without epithelial cell; maintaining the living rate of frozen cell between 93. 2% and 96. 7%; fitting for large scale of culturing; providing new method to clone cell and main material to manufacture vaccine.

Description

'Jingning ' chicken embryo fibroblast series and cultural method thereof

Technical field

The present invention relates to a kind of clone and cultural method thereof, specifically utilize the Jingning Embryo Gallus domesticus to carry out first culture, go down to posterity and cultivate the high motility rate of acquisition, high purity 'Jingning ' chicken embryo fibroblast series, belong to the cytobiology field.

Background technology

Along with improving constantly of development of science and technology and people's material life demand, livestock industry is just produced towards the direction fast transition and the development of high yield, high-quality, degeneration-resistant, disease-resistant and intensification.In most of developed countries, because people are cause such as economic pursuit interests too, the livestock and poultry that breed in a large number are high kind of minority economic worth and cross-fertilize seed, thereby, caused and a lot of output is not high, the local variety of bad, degeneration-resistant and disease resistance difference endangered, even extinction.Though relevant international organization, government department and experts and scholars quite pay attention to the preservation and the management work of livestock and poultry genetic resources, livestock and poultry germ plasm resource is still in a large amount of forfeitures, and genetic diversity has suffered unprecedented destruction, and criticality increasingly sharpens.

Nineteen ninety-five, the statistic data of FAO shows: area, the Central Africa registration livestock and poultry on record at Sub-Sahara have 738 kinds approximately, and nearly 15% is livestock and poultry resource in imminent danger.The geographic form in Africa is very severe, and the livestock and poultry genetic resources has suffered from destroying, from nineteen ninety-five so far, be in endangered poultry and risen to 34% by 20%.The Asian-Pacific area: have the livestock and poultry resource in the whole world 1/5,1251 livestock and poultry species are registered, and in the kind that is recorded, have 10% to be resource in imminent danger approximately according to estimates.In the period of nineteen ninety-five to 1999, the endangered poultry in Asia rises to 37% by 32%.This situation is even more serious in Eastern Europe, in several years of nineteen ninety-five to 1999 year, is in endangered poultry and has risen to 76% by 65%.Latin America: existing 20% kind considered to be in imminent danger.The bird that is in Critical Condition by 5% of nineteen ninety-five sharply risen to 1999 45%.Area, the Near East: the intensification of production and mechanize have caused the endangered of a lot of livestock and poultry species.In 571 kinds that are registered, there are 44 even be considered to be in Critical Condition more.The North America: lasting production intensification, make the kind only rely on minority aborning satisfy the demand of foodstuff production, therefore increased the weight of the multifarious criticality of livestock and poultry genetic resources, in 259 kinds that are recorded, have 35% endangered.According to 2000 Food and Argriculture OrganizationFAO (FAO) with United Nations Environment Programme (UNEP); present global livestock and poultry species is being lost with the speed that reduces by 2 kinds weekly; in by 2576 livestock and poultry species that FAO write down; almost half is considered to livestock and poultry resource in imminent danger; there are 1350 kinds to face extinction approximately; make world livestock and poultry germ plasm resource more and more narrower general layout occur, therefore save and protect livestock and poultry species in imminent danger to become the task of top priority.

China's Production of Livestock and Poultry power type is abundant, and its genetic resources has species and various in style, origin and widely distributed, and characteristics such as ecotype is various, and the shared ratio of China's local variety is higher than other countries far away.China livestock and poultry have the characteristic strong to the eqpidemic disease resistibility, the excellent genes with various resistances and economic characters.

But, in the last few years, because all multifactor influences such as a large amount of introductions of the continuous deterioration of ecotope, external livestock and poultry species and intensive culture cause the extinction of a lot of good local livestock and poultry species of China or are in endangered state.Nearly 20 years, 41.9% local variety colony quantity had decline in various degree.The middle and later periods eighties, the supporting system of chicken replaced local chicken breed and the new kind of cultivating again, caused China's domestic animal germ plasm resource to be subjected to havoc.The livestock and poultry genetic resources diversity situation of China is also also pessimistic, and the trend of continuous deterioration is arranged.The crisis of livestock and poultry germ plasm resource means the crisis of the entrained gene of livestock and poultry germplasm, or even the eternal disappearance of gene, means multifarious decline of livestock and poultry genetic resources and forfeiture.The extinction of kind means that cell and molecular biology carry out the eternal forfeiture of the basic material of scientific research to it, under if the germ plasm resource of these kinds had not been preserved before extinction by any way, not only lost its genetic resources forever, and make scientists will become mystery and dream forever for the exploration of the cell of their many unknowns and molecular biology mechanism and the hope that makes it to reproduce by somatic cell clone technique, also will be simultaneously the massive losses of the theoretical treasure-house of world's genetic resources legacy and life science.Therefore, in today of global plunder Biological resources and protection species diversity, from China livestock and poultry germ plasm resource preserve actual, the mode of important by making up, in imminent danger livestock and poultry species colony cell bank is preserved its genetic resources, and is significant.

In recent years, because cultured cell in vitro has huge scientific value and using value in all many-sides such as genetic resources preservation and life sciences, caused that national governments pay much attention to.Except that the U.S., Osaka, Japan fermentation institute (IFO), German culture collection institute (DSM) etc. are all enlarging the preservation scope gradually, list cell culture in the preservation object.

At present, the development of cryogenic freezing preservation technology and perfect, make any vitro culture thing on the basis that keeps its vigor, to be preserved indefinitely, therefore, the major issue that is undoubtedly present stage cytobiology area research for a large amount of cultivations of zooblast with solution germ plasm resource problem.

The Jingning chicken is the local breeding poultry of ovum meat dual-purpose type that originates in Jingning, China Gansu.Its head is held high, and tail feathers is towering, and the chest muscle prosperity is carried on the back wide and long.Cock mostly is rose comb, and the reddish brown and red sauce of hair color is main, and primaries, main tail feather are black, beautiful appearance; Hen is little delicate and pretty, and yellow accounts for major part, and the ratio that numb look accounts for is quite a few, and its delicious meat delicacy adopts peasant household to disperse to raise at present, and the mountain area is to herd.But, because the pollution and the deterioration of physical environment in recent decades, the germ plasm resource of Jingning chicken is seriously deficient, become extinct in most areas, also only also there is limited number in the small portion area, and many-sided technical problem that present artificial breeding is faced causes it to breed poor effect, and China Jingning chicken faces endangered danger.More because the scarcity of its germ plasm resource makes fields such as biology, medical science also become blank for the research of Jingning chicken.Therefore, treasure the fowl kind for protecting country, the method for an optimum is to the large scale culturing of Jingning chick fibroblast and in addition preservation.

Along with development of biology, press for large-scale cell cultures at present, so that obtain a large amount of useful cell expression products.But; because Jingning chick fibroblast poor growth is very responsive to culture environment, if directly few from former foster its cell quantity that is obtained of being commissioned to train of tissue block; cell type is many and mix; as often mixing in the inoblast epithelial cell is arranged, along with the prolongation of incubation time, difference appears in the intercellular growth time limit of the contained all kinds of culture; the cell type that has is the accelerates multiplication growth through adapting to growth phase; and other cells are perhaps dead, perhaps arrive dead through progressively degenerating.And first-generation cell culture technology key problem is to be difficult to industrialization or perhaps large-scale production: the one, and can not the mass preparation product when explained hereafter; The 2nd, non-batch process causes the unhomogeneity of quality product easily; The 3rd, be difficult to producing and quality control with criticizing.And the employing vial leaves standstill or the cultural method of revolving bottle, also can not satisfy required cell quantity and secretory product thereof.Can obtain high-density cells though use more reactor to cultivate at present, but the expansion scale is difficult, only is suitable for a small amount of, costly cell cultures.

At present, normal Epstein-Barr virus transformation technology, adherent culture method and the collagenase digesting technology of adopting carried out a large amount of cultivations of bird kind cell in vitro, but comparative test result shows: the Epstein-Barr virus transformation technology is cultivated for the cell in vitro of bird kind and is inapplicable, its cultured cells motility rate and purity all can not reach required standard, and the Epstein-Barr virus transformation technology is better than the bird cell cultures for the cell cultures effect of human and primate; The primary cell that collagenase digesting technology and adherent culture method obtain inoculation after going down to posterity respectively, the cell colony doubling time, (PDT) was respectively 35.9h and 48h.But the former operation steps is numerous and diverse, is not suitable for the cultivation of external extensiveization cell, and the latter is simple, is suitable for the cultivation of external extensiveization cell.Therefore set up clone to adopt the latter for good.For the preservation of bird cell, cell purity and activity all have higher requirement, and at present, there are many drawbacks in the adherent culture method, and be single as method, cost is too high, cytoactive and purity is on the low side, cell cryopreservation DAR rate is high excessively.Therefore, being badly in need of now making the Jingning chick fibroblast reach the highly purified standard of high cell motility rate to its method improvement has obtained good preservation and has continued.

Summary of the invention

The purpose of this invention is to provide the 'Jingning ' chicken embryo fibroblast series of a kind of high cytoactive, high cell purity, its preserving number is: CGMCC No.1878.

Another object of the present invention is to provide the cultural method of above-mentioned clone.

For achieving the above object, the present invention takes following technical scheme:

A kind of high motility rate, high purity 'Jingning ' chicken embryo fibroblast series, and at China Committee for Culture Collection of Microorganisms common micro-organisms center, CGMCC (address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City; Postcode: 100080; Phone: 010-62542758), preservation day: on December 1st, 2006, preserving number: CGMCC No.1878, the classification called after 'Jingning ' chicken embryo fibroblast series of suggestion.This 'Jingning ' chicken embryo fibroblast series has following feature: promptly have cell to grow behind the embryonic tissue piece inoculation 6h of Jingning chicken, can stick a bottle wall in 1～2 day.Visible cell is transparent under the inverted microscope, and the kytoplasm projection is plentiful, is spindle shape, karyon ellipse, placed in the middle, and kernel is clear.After cell growth in flakes, cell is spindle shape, flamboyancy, and cellular form reaches unanimity, for monokaryon becomes fiber-like.Simultaneously, cellularity is stable, and propagation is very fast.

The cultural method of above-mentioned high motility rate, high purity 'Jingning ' chicken embryo fibroblast series, this method comprises the steps:

(1) first culture: will cut into 0.5～1.5mm with after the PBS rinsing 3～4 times except that the Jingning Embryo Gallus domesticus of decerebrate, eye, four limbs, internal organ ³Tissue block; Tissue block is moved into culturing bottle, be inverted culturing bottle, and at 37.5 ℃, 5%CO ₂CO ₂Cultivate 3～4h in the incubator; The complete adherent back of tissue block adds substratum 6～10ml, medium component: the MEM+10% foetal calf serum, continue to cultivate 10～12h;

(2) cultivation of going down to posterity: the nutrient solution in reject step (1) culturing bottle behind residue in the PBS washing culturing bottle 2 times, stops digesting with adding full nutrient solution (MEM of 10%FBS) 6～10ml behind the tryptic digestion; Postdigestive cell average mark is packed in 2 culturing bottles, puts into 37.5 ℃, 5%CO ₂CO ₂Continue to be cultured to frozen preceding 24h in the incubator;

(3) cell cryopreservation:

A, the interior nutrient solution of frozen preceding 24h reject culturing bottle are also changed fresh medium 6～10ml, the nutrient solution composition: the MEM+10% foetal calf serum, continue to cultivate 24h;

B, use the tryptic digestion culturing cell, add full nutrient solution 6～10ml termination reaction then;

C, usefulness red blood cell count(RBC) plate calculate frozen preceding total cellular score;

Centrifugal 6～the 10min of D, collection: 1000rpm removes supernatant liquor, adds the frozen storing liquid 1ml of 4 ℃ of precoolings, blows and beats gently with suction pipe behind the mixing to make cell resuspended; Frozen storing liquid composition: 10%DMSO+50% foetal calf serum+40%MEM;

E, the culture packing gone in the frozen pipe of sterilization to seal;

F, pre-freeze: with frozen pipe pack into the cooling box in, be put in 4 ℃ of 20～30min, place-70 ℃ then more than the pre-freeze 4h;

G, frozen: propose frozen pipe and put into the liquid nitrogen cabinet, promptly finish cell cryopreservation.

The cultural method of above-mentioned high motility rate, high purity 'Jingning ' chicken embryo fibroblast series, wherein, Jingning Embryo Gallus domesticus 6 ages in days described in the step (1) are best.

The cultural method of above-mentioned high motility rate, high purity 'Jingning ' chicken embryo fibroblast series, wherein, trysinization concrete steps described in step (2) and the step (3) are: add 0.25% trypsinase, 1.5～2.5ml in culturing bottle, upset digestion 30～60s in the back inversion culturing bottle is preheated to 37 ℃ in incubator after.

The cultural method of above-mentioned high motility rate, high purity 'Jingning ' chicken embryo fibroblast series, wherein, can carry out cell recovery by the cell that step (3) is frozen according to actual needs, concrete steps are: frozen pipe is taken out from liquid nitrogen insert in 42 ℃ of water-baths, after rocking 1min continuously, cell moved in the culturing bottle be added with nutrient solution (MEM+10% foetal calf serum) piping and druming evenly, place and contain 37.5 ℃ of 5%CO ₂Incubator in can continue the cultivation of going down to posterity after continue cultivating 10～12h.

Advantage of the present invention and benefit: the present invention can make the inoblast of turning out not have impurity cells such as epithelial cell for the improvement adjustment of adherent culture method and culture medium culturing liquid composition, and cell purity has been compared significantly in prior art and improved; The cell that makes recovery for the improvement of frozen condition with frozen before compare, cell type still is an inoblast, the cellular form and the speed of growth do not change, the freeze-stored cell steady quality, and the motility rate of cell can reach and maintain between 93.2%～96.7% behind the cell cryopreservation, and the passage growth is stable, compares with existing culture technique cultured cells to be significantly increased, and is fit to large scale culturing.

Improve a lot with the Jingning chick embryo fibroblast culture efficiency and the quality of enzyme digestion than adherent culture method, utilizing enzyme digestion to cultivate among the contrast experiment of Jingning chick embryo fibroblast finds, the difference that primary cell growth conditions and adherent culture are sent out is very little, find promptly about 2d that cavity appears in cell but go down to posterity, and serious day by day with the increase cell vacuolation of incubation time, can't obtain the Jingning chick embryo fibroblast of state of health.Therefore, utilize the adherent culture method to cultivate the Jingning chick embryo fibroblast and have remarkable advantages.Cultured cells fast growth of the present invention, surviving rate height, exogenous gene expression rate height, cell confluency and apoptosis rate are low, applied range.

For the adjustment of substratum and the improvement of cultural method, cost is significantly reduced.The present invention has remedied the unfavorable situation of existing fowl somatocyte preservation condition in each side such as methods, and the genetic resources that the motility rate of 'Jingning ' chicken embryo fibroblast series and the lifting of purity also make Jingning important, in imminent danger chicken breed is able to prolonged preservation with the form of cultured cell in vitro.

Because the scarcity of Jingning chicken germ plasm resource, making becomes blank for the research of Jingning chicken in each field.And high cell motility rate of the present invention and highly purified 'Jingning ' chicken embryo fibroblast series can provide a large amount of high quality material for life sciences such as medical science, cell and molecular biology; And can be used as the donorcells of fowl somatic cell clone breeding; On agricultural, can enrich the means that local fowl breed improvement and local good fowl kind guarantor plant; Can also conduct be the main raw material(s) of production of vaccine and the feeder layer of liver cell culture; The usefulness that can be used for apoptosis and cytogamy research simultaneously.

The present invention will be further described below in conjunction with accompanying drawing and preferred forms, so that the public has whole to summary of the invention and understand fully, and is not qualification to protection domain of the present invention.Aforementioned part fully discloses the protection domain that the present invention can implement, and therefore allly any well known in the artly is equal to replacement according to what the disclosure of invention was carried out, all belongs to infringement of the present invention.

Description of drawings

Fig. 1 is that the Jingning CEO is for microcytoscope figure below;

Fig. 2 for the Jingning chicken go down to posterity before microcytoscope figure below;

Fig. 3 is Jingning chicken subculture I microcytoscope figure below;

Fig. 4 is Jingning chicken embryo subculture II microcytoscope figure below;

Fig. 5 is by the inoblast of mycoplasma contamination contrast figure;

Fig. 6 is a Jingning chick embryo fibroblast detection of mycoplasma negative findings diagram;

Fig. 7 is a Jingning chick embryo fibroblast growth curve chart;

Fig. 8 is a Jingning chick embryo fibroblast caryogram diagram;

Fig. 9 is Jingning chick embryo fibroblast lactic dehydrogenase isozyme electrophoresis diagram (two swimming lanes in left side are the lactic dehydrogenase isozyme that white eared pheasant, two swimming lanes in right side are the Jingning chicken);

Figure 10 is Jingning chick embryo fibroblast malate dehydrogenase (malic acid dehydrogenase) electrophoresis diagram (two swimming lanes in right side are oxysuccinic acid acid isozyme white eared pheasant, that two swimming lanes in left side are the Jingning chicken);

Figure 11 is that foreign gene is expressed 24h diagram (transfection of pEGFP-N3 fluorescin plasmid) in the chick embryo fibroblast of Jingning;

Figure 12 is that foreign gene is expressed 48h diagram (transfection of pEGFP-N3 fluorescin plasmid) in the chick embryo fibroblast of Jingning;

Figure 13 is that foreign gene is expressed 48h diagram (transfection of pDsRed1-N1 fluorescin plasmid) in the chick embryo fibroblast of Jingning;

Figure 14 is that foreign gene is expressed 48h diagram (transfection of pEYFP-N1 fluorescin plasmid) in the chick embryo fibroblast of Jingning;

Painted Jingning, Figure 15 Yihong chicken cell (400 *);

The painted White Yak in Tianzhu cell of Figure 16 DAPI (400 *);

Figure 17 White Yak in Tianzhu and Jingning chicken cell merge (1000 *);

Figure 18 White Yak in Tianzhu and the Jingning a certain site of chicken fused cell enlarged view (400 *);

Figure 19 Jingning chicken cell self merges (400 *);

Apoptosis detects (200 *) to chicken recovery back, Figure 20 Jingning.

Embodiment

Used key instrument and reagent source among the embodiment:

Chicken embryo source, Jingning: Jingning egg embryo picks up from the Jingning County, Gansu Province.

(1) key instrument equipment

1, CO ₂Incubator: Heraeus BB16UV;

2, DL-CJ-2N high-performance aseptic experiment platform: Harbin;

3, IX-71 inverted phase contrast microscope, CX31 biomicroscope, IX-71 are inverted and are differed fluorescent microscope: Olympus;

4, TDL-40B low speed centrifuge: CENTERIGUGE;

5, your Superpure water machine: Pall-pruelab_plus quite;

6, electronic pipettor: German Accu-jet;

7 ,-70 ℃ cryogenic refrigerator: U.S. kelvinator;

8, laser scanning co-focusing microscope: Nikon TE-2000-E;

9, liquid nitrogen container: East Asia, Sichuan YES-50B-125F;

10, electrophoresis apparatus DYY-6C and electrophoresis chamber DYC-24A: Beijing Liu Yichang;

(2) main agents

1, MEM (contain Earle ' s salt, L-glutamine, non-essential amino acid, do not contain hydrocarbonate): Gibco company;

2, liposome Lipofectin:Gibco company;

3, carrier pEGEP-N3:Clontech;

4, trypsin Trypsin is 1: 250), Jim Sa (Giemsa): Amresco;

5, foetal calf serum (Defined FBS): superfine foetal calf serum (Defined FBS), Hyclone;

6, trypan blue (Trypan Blue), DMSO, colchicine (Colchicine), EDTA, Triton (Triton) X-100＞99%, TEMED, Bis (methylene diacrylamide) 98%, ammonium persulphate (AmmoniumPersulfate), PMS, NAD, tetrazolium bromide (Thiazolyl Blue): Sigma;

7, glycerine analytical pure Hua Lvyuan;

8、Tris：Promega；

9, acrylamide＞99% (Acr), glycine＞99% (Clycine): NOVON;

10、PEG：Mylab；

Inoblast is cultivated agents useful for same:

11, MEM nutrient solution: the MEM of ultrapure tri-distilled water dissolving 9.6g, the NaHCO of 2.2g ₃, add water and be settled to 1L, after pH is 7.2～7.4,0.22 μ m membrane filtration degerming, the packing of 500ml/ bottle, 4 ℃ of storages;

12, full nutrient solution: adding final concentration in the MEM nutrient solution is the FBS of 10% (volume ratio), after the 0.22 μ m membrane filtration degerming, and the packing of 500ml/ bottle, 4 ℃ of storages.

13, two anti-stock solutions: 4 of the Streptomycin sulphates of 1,000,000 IU, 5 in the penicillin of 800,000 IU are dissolved in the 400ml sterilization deionized water.

14,1 * PBS damping fluid: NaCl4.00g, KCl0.10g, Na ₂HPO ₄12H ₂O1.45g, KH ₂PO ₄0.10g, adding tri-distilled water and be settled to 500ml, pH is 7.2, autoclaving seals back 4 ℃ of storages.

15,0.25% (mass volume ratio) trypsin solution: 1.25g trypsinase dry powder is dissolved among the PBS, is settled to 500ml, after pH is 7.2～7.4,0.22 μ m membrane filtration degerming, and packing ,-20 ℃ of storages.

16,0.9% (mass ratio) physiological saline: 0.45g NaCl is dissolved in the 500ml tri-distilled water, autoclaving 30min.

17, cells frozen storing liquid: 50ml DMSO is added in the full nutrient solution of 450ml (the full nutrient solution that contains volume ratio 15%FBS),,, be stored in-20 ℃ of refrigerators with the packing of 100ml/ bottle with 0.22 μ m membrane filtration degerming.

The microorganism detection agents useful for same:

18, soybean Tryptones: 3g soybean Tryptones, add the 100ml tri-distilled water, transfer pH to 7.3, autoclaving 15～20min divides to install in the Glass tubing of 15mm * 150mm.

19, wort: the 2g wort adds the 100ml tri-distilled water, transfers pH to 3～4, and autoclaving 15～20min divides to install in the Glass tubing of 15mm * 150mm.

20, DAPI dye liquor: tri-distilled water preparation 1mg/ml DAPI storage liquid, the PBS dilution is 0.5～1mg/ml.

21, mounting liquid: the citric acid of 22.2ml 0.1M, the Na of 27.8ml 0.2M ₂HPO ₄Be dissolved in the 50ml glycerine, NaOH regulates pH to 5.5, is stored in 4 ℃.

22, stationary liquid: Glacial acetic acid and methyl alcohol are with 1: 3 ratio of volume ratio (matching while using).

Chromosome specimen prepares agents useful for same:

23, colchicine stock solution: the 10mg colchicine is dissolved in the 10ml tri-distilled water.

24, diluent: tri-distilled water is 100 μ g/ml with 10 times of stock solution dilutions to final concentration.

25, hypotonic KCl solution: the KCl of 0.5g is dissolved in the 100ml tri-distilled water.

26, stationary liquid: 1: 3 by volume ratio of Glacial acetic acid and methyl alcohol preparation.

27, Giemsa staining fluid

Stoste: 1g Giemsa is dyed powder pour mortar into, add several glycerine, be ground in mortar till the no particle, glycerol adding is to 33ml then, be placed on 56 ℃ of insulation can 2h after, add 45ml methyl alcohol and stir, store standby in the brown bottle.

Working fluid: phosphoric acid buffer is 10ml with 10 times of Giemsa stoste dilutions to final concentration.

28, the preparation of phosphoric acid buffer: KH ₂PO ₄0.34g add 100ml deionized water (transferring pH 6.8) with NaOH

Liposome transfection cell agents useful for same:

29, plasmid DNA (DsRed, pEYFP-N1 and pEGFP-N3), liposome Lipofectamine does not contain the MEM nutrient solution of serum, contains the MEM nutrient solution of serum, the PBS of pH 7.4.

The Isozyme Analysis agents useful for same:

30,0.9% (mass ratio) NaCl-0.06mmol EDTA solution: take by weighing 0.9g NaCl and 0.0223gEDTA respectively and be dissolved in the 100ml tri-distilled water and get final product.

31, protein extract:, it is stored in 4 ℃ of refrigerators preserve with promptly getting protein extract in 5ml TritonX-100 adding 75ml 0.9% (mass ratio) the NaCl-0.06mmolEDTA solution.TritonX-100: 0.9% (mass ratio) NaCl-0.06mmol EDTA solution=1: 15.

32,40% (mass volume ratio) sucrose solution: 40g sucrose is dissolved in the 100ml water.

33, the preparation of sol solution

A liquid: 1mol/L HCl 48.0ml, Tris 36.6g, TEMED 0.23ml transfers to pH 8.9 with concentrated hydrochloric acid

B liquid: Acr 40.0g, Bis 2g

C liquid: ammonium persulphate 0.14g (now with the current)

D liquid: 1mol/L HCl 48.0ml, Tris 5.98g, TEMED 0.46ml transfers to pH 6.7 with concentrated hydrochloric acid

E liquid: Acr 10.0g, Bis 2.5g

F liquid: riboflavin 4.0mg

G liquid: sucrose 40.0g

34, polyacrylamide gelating soln concentration sees Table 1

Table 1

35, the configuration of electrode buffer: 6g Tris, the 28.8g glycine is dissolved in 1000ml distilled water, and pH transfers to 8.7

36, electrophoresis indicator: scale takes by weighing the 0.25g tetrabromophenol sulfonphthalein and 40g sucrose is dissolved in the 100ml water respectively.

37, LDH staining fluid preparation (1h preparation before the dyeing)

Calcium lactate 100mg+0.05M Tris-HCl (pH 8.0) 20ml

Tetrazolium bromide 5mg+ tri-distilled water 1.0ml

PMS 2.5mg+ tri-distilled water 1.0ml

Nadide (NAD) 10mg

Before the dyeing, after above insulation reagent mix, pour in 20ml 2% (volume ratio) agar-agar soln mixing into.

38, MDH staining fluid preparation (1h preparation before the dyeing)

DL-oxysuccinic acid 350mg is dissolved among the 25ml 0.1M Tris-HCl (pH 8.0)

Tetrazolium bromide 15mg+ tri-distilled water 1.5ml

PMS 5mg+ tri-distilled water 1.0ml

Nadide (NAD) 10mg

After above mixing, pour in 25ml 2% (volume ratio) agar-agar soln.

39, the preparation of stationary liquid: ethanol 10ml, acetate 40ml, glycerine 20ml adds water to 80ml.

40, tetrabromophenol sulfonphthalein solution must be prepared: take by weighing the 0.25g tetrabromophenol sulfonphthalein respectively and 40g sucrose is dissolved in the 100ml water.

The cytogamy agents useful for same:

41, GKN solution: NaCl 8g, KCl 0.4g, Na ₂HPO ₄12H ₂O 3.58g, NaH ₂PO ₄0.68g, glucose 2g, phenol red 0.01g is dissolved in the 1000ml distilled water.

42,50% (mass volume ratio) PEG solution: the PEG (WM=4000) that takes by weighing 50g puts into beaker, and boiling water bath heating makes it fusing, and is to be cooled during to 50 ℃, add to be preheated to 50 ℃ GKN solution 100ml, mixing, put 37 ℃ standby.

43,0.5mol/L CaCl ₂The anhydrous CaCl of solution: 5.5495g ₂Be dissolved in the 100ml tri-distilled water, final concentration is 0.5mol/L.

44, DAPI dye liquor: tri-distilled water preparation 1mg/ml DAPI storage liquid, the PBS dilution is 0.5～1mg/ml.

45, eosin stain (test kit)

Apoptosis detects agents useful for same:

46, stationary liquid

Glacial acetic acid and anhydrous methanol be with 1: 3 mixed of volume ratio, 4 ℃ of storages standby (matching while using).

47, DAPI dye liquor: tri-distilled water preparation 1mg/ml DAPI storage liquid, the PBS dilution is 0.5～1mg/ml.

48, the preparation of mounting liquid: with 22.2ml 0.1mol/L citric acid and 27.8ml 0.2mol/L Na ₂HPO ₄Join in the 50ml glycerine, transfer pH to 5.5 with NaOH, 4 ℃ of storages are standby.

The cultivation of embodiment 1 high cell motility rate high purity 'Jingning ' chicken embryo fibroblast series

(1) first culture: under the aseptic condition,, carefully take out the embryo with the ophthalmology tweezer with Jingning chicken broken big end of eggshell of instar embryo on the 6th, place in the little culture dish, remove embryo's brain, eye, four limbs, internal organ, put into the culture dish rinsing 3～4 times that PBS is housed, to remove surperficial blood stains; Tissue is cut into 0.5～1.5mm ³Tissue block; Tissue block is moved in the culturing bottle respectively be uniformly dispersed with moving the sample device, be inverted culturing bottle, and at 37.5 ℃, 5%CO ₂CO ₂Cultivate 3～4h in the incubator; The complete adherent back of tissue block adds substratum 8ml, and the superfine foetal calf serum of medium component: MEM+10% continues to cultivate 10～12h;

(2) cultivation of going down to posterity: the nutrient solution in reject step (1) culturing bottle, behind residue in the PBS washing culturing bottle 2 times, to remove remaining serum and tissue block and the dead cell that comes off, in culturing bottle, add 0.25% trypsinase 2ml, be inverted the digestion 50s that overturns culturing bottle is preheated to 37 ℃ in incubator after, observation of cell matter retraction under inverted microscope when the intercellular substance increases, adds full nutrient solution (MEM of 10%FBS) 8ml and stops digestion; Postdigestive cell average mark is packed in 2 culturing bottles, puts into 37.5 ℃, 5%CO ₂CO ₂Continue to be cultured to frozen preceding 24h in the incubator;

(3) cell cryopreservation:

A, the interior nutrient solution of extremely frozen preceding 24h reject culturing bottle are also changed fresh medium 8ml, and the superfine foetal calf serum of nutrient solution composition: MEM+10% continues cultivation 24h;

B, add 0.25% trypsinase 2ml in culturing bottle, upset digestion 50s after being inverted culturing bottle being preheated to 37 ℃ in incubator adds full nutrient solution 8ml termination reaction;

The centrifugal 8min of D, collection: 1000rpm removes supernatant liquor, adds the frozen storing liquid 1ml of 4 ℃ of precoolings, blows and beats gently with suction pipe behind the mixing to make cell resuspended; Frozen storing liquid composition: 10%DMSO+50% foetal calf serum+40%MEM;

E, the culture packing gone in the frozen pipe of sterilization and tightly seal, indicate date, kind, cell title, cultivate algebraically;

F, pre-freeze: with frozen pipe pack into the cooling box in, be put in 4 ℃ of 20～30min, then pre-freeze 10～12h (more than the 4h) in-70 ℃ of refrigerators;

(4) cell recovery: frozen pipe is taken out from liquid nitrogen, inserts rapidly in 42 ℃ of water-baths, rock 1 fen kind continuously after, seeing surplusly has little ice group to become the big h of soybean grain to put into Bechtop; Cell moved in the culturing bottle be added with nutrient solution (the superfine foetal calf serum of MEM+10%) piping and druming gently evenly, place and contain 37.5 ℃ of 5%CO ₂CO ₂Can continue the cultivation of going down to posterity after continuing in the incubator to cultivate 10～12h.

(5) purifying cells: utilize trysinization to carry out the purifying inoblast.From the clone morphological observation result who builds up, to support and go down to posterity in early days when cultivating former being commissioned to train, epithelial cell and inoblast occur simultaneously, mix growth, and it is in blocks to mix the grown cell subregion.Epithelial cell is a Polygons, with inoblast a great difference is arranged on the form.Because epithelial cell is different to the trypsinase tolerance with inoblast, when the digestion culturing cell, often be that inoblast takes off wall earlier, and the back of going down to posterity is adherent fast, adheres to fast.And other most of epithelial cell can not adhere to or adhere to instability at short notice, vibration is promptly floated slightly, need the support of growth matrix such as collagen or other extracellular matrix components etc., therefore, utilize this difference, through enzyme digestion with after adherent method handled for 2～3 generations repeatedly, purifying inoblast fully.Cell is typical fusiformis, and there is oval nuclear in central authorities, and that cell is when growth is radial, flamboyancy or swirl shape traveling.Cell purity reaches more than 99%.

The detection of embodiment 2 'Jingning ' chicken embryo fibroblast series biological characteristicses

The 'Jingning ' chicken embryo fibroblast series that embodiment 1 is cultivated carries out the biological characteristics inspection, and method and conclusion are as follows.

One, morphological observation

Method: cell is in the vitro culture process, and pair cell carries out routine examination, and whether observation of cell growth conditions, nutrient solution colour-change situation and cell pollute.

Conclusion: shown in Fig. 1 (Jingning chicken primary cell), Fig. 2 (Jingning chicken go down to posterity preceding cell), Fig. 3 (Jingning chicken subculture I cell), Fig. 4 (Jingning chicken subculture II cell), when cell growth state is good, its form is that typical one-tenth is fibrous, presents fusiformis or sealene triangle.The overall picture of pair cell colony is observed, and that institute's cultured cells all is is radial, flamboyancy or whirlpool shape tendency, proves that the cell healthy growth is vigorous.

Two, microorganism detection

1, bacterium, fungi detect

Method:

(1) get the freeze-stored cell of total freeze-stored cell ampoule 0.5%, be dissolved in respectively in the 8ml nonreactive substratum, behind the mixing with cell suspension with the centrifugal 20min of 2000g, and repeat twice, to eliminate antibiotic influence.Be resuspended in again in the nutrient solution of 2ml antibiotic-free.

(2) cell is inoculated in Trypsin beans peptone and the malt extract medium with 0.5ml, places the incubator of 37 ℃ and 26 ℃ to cultivate for two weeks respectively.

(3) establishing contrast is:

A. positive control: subtilis and Candida albicans bacterium are inoculated into respectively in Trypsin beans peptone and the malt extract medium to be cultivated, and places the incubator of 37 ℃ and 26 ℃ to cultivate for two weeks.

B. negative control: Trypsin beans peptone substratum and malt extract medium place the incubator of 37 ℃ and 26 ℃ to cultivate for two weeks respectively.

Conclusion: detect soybean Tryptones nutrient solution and wort nutrient solution that cell visual inspection inoculation has the Jingning chick fibroblast, all present limpid transparence, place microscopically to observe in test tube, remove the rounded transparence bright spot of zooblast and swim in the nutrient solution, do not have other foreign matter.In the whole process of proof and cell recovery frozen in cell cultures, cell is not by bacterium, fungal contamination.

2, virus detects

Method:

(1) inoculates cell culture to be detected, be cultured to fine and close individual layer with the nutrient solution of antibiotic-free.

(2) gather fresh anticoagulant heparin whole blood (chicken blood, guinea pig blood), the centrifugal 10min of 1000rpm abandons supernatant.Sedimentary red corpuscle suspends with 0.9%NaCl solution with PBS washing 3 times, makes the red cell suspension of 0.5% (V/V).

(3) cell reject nutrient solution to be checked washes monolayer cell 2～3 times with 0.9%NaCl solution.

(4) add the freshly prepared chicken of 0.5ml, guinea-pig red blood cell suspension respectively in cell bottle to be checked, place 20min in 4 ℃.

(5) observe red cell agglutination and adsorption phenomena, do not present HA cell, need repeat (2)～

(4) operation.

Conclusion:

Because some virus surface has hemagglutinin, can hemagglutination occur in conjunction with the red corpuscle of some species.The red corpuscle of chicken, cavy can be adsorbed in its surface by the cell of these virus infectiones.Detected result: the conventional Jingning chick embryo fibroblast of cultivating under the condition of antibiotic-free, phase microscope is observed, and the red corpuscle of chicken is ellipticity and is suspended in the inoblast top, agglutination phenomenon do not occur; Agglutination phenomenon does not appear in the rounded top that is suspended in of the red corpuscle of cavy yet.Detected result is negative, proves that this cultural method cultured cells does not cause cell injury because of virus in culturing process.

3, detection of mycoplasma

Method:

(1) sample: inoblast is made cell suspension, and adjusting cell concn is 1 * 10 ⁵Individual/ml.

(2) cultivate: tested cell goes down to posterity to cultivate in not containing antibiotic nutrient solution and (is not less than 3 times) more than 3 times, goes down to posterity the last time to cultivate to breed the interim bright nutrient solution that renews, and continues to cultivate 2～3d.

(3) inoculation: the negative control ware adds cell nutrient solution MEM 2ml; Add sample 2ml to be checked in the ware to be checked, treat from bottle, to take out before the cover plate culturing cell converges (converge fully as cell, influence is to the observation of mycoplasma).

(4) rinsing: the cell cover plate is placed the dish ware, use the PBS rinsing, cold wind dries up.

(5) fixing: soak cover glass with stationary liquid, behind the fixed cell 10min, repeating step (4).

(6) dyeing: will prepare Hoechst 33258 dye liquors and be added drop-wise on the cell that fixes, 30min dyes under the room temperature.

(7) rinsing: embathe cover glass 3 times after the dyeing with PBS, each 3～5min, cold wind dries up.

(8) film-making a: PBS who contains 1% glycerine is added to cell surface after the dyeing, will has the cover glass of cell to face down and cover on the slide glass.

(9) observe: with 100 * to 400 * fluorescence microscope, open light source 10min after, excite with 330～380nm purple fluorescence, whether observation of cell nuclear is outer has the fluorescence of blue-fluorescence point or thread point to produce.

Conclusion:

Hoechst 33258 fluorescence dyes can see through the complete cell of cell membrane, embed in the nucleus DNA, make it to send bright blue-fluorescence.Also contain DNA in the mycoplasma, also can be painted, under the fluorescence microscopy Electronic Speculum, observe and can see blue-fluorescence.Therefore, in positive control,, around reaching, cell surface also can see blue point-like or thread fluorescence except that the nucleus position has the blue-fluorescence.And mycoplasma DNA fluorescent dye particle and thread point are arranged simultaneously at cell surface, as shown in Figure 5.Detected result shows: as shown in Figure 6, after the chick embryo fibroblast film-making of vitro culture Jingning, under inverted fluorescence microscope, purple fluorescence with 380nm excites, can find that visible nucleus surface is smooth sample in the whole visual field, nucleus is round shape or ellipticity, and the DNA in the nucleus sends blue-fluorescence, and cell peripheral does not have thread or the particulate state point.

Three, cell growth curve is drawn

Method:

(1) suspension preparation

Get growth conditions good cell to be measured, when increasing to approaching converging, make cell suspension with the ordinary method peptic cell, and counting.

(2) inoculation

In 24 holes of culture plate, inoculate the cell of equal amts respectively, be generally (1～2) * 10 ⁴Individual cell/ml continues to cultivate in 37 ℃ of CO2 incubators.

(3) count detection

Count from inoculation time, the cell density in 24h counts 3 holes calculates total cellular score with blood counting chamber, calculates mean value, gets the mean value in 3 holes, so to the 8th group of end.

(4) draw

With incubation time (d) is that X-coordinate, cell density are ordinate zou, and the result is drawn on graph paper, promptly gets the growth curve of culturing cell.

Conclusion:

By the Jingning chicken cultured cell in vitro of being cultivated is prepared cell suspension with ordinary method, counting, inoculate 24 well culture plates, every hole 1ml cell suspension, the cell that is inoculated in 24 well culture plates is carried out regularly numeration in continuous 7 days, write down each time cell density, draw and form the culturing cell growth curve as shown in Figure 7, the inoculation back increases since the 2nd day cell count, entered logarithmic phase on the 3rd day, the 6th day cell poor growth, cell general trend in vegetative period is all S-type, and all arranged behind the cell inoculation latent period of 24h, this phase is the laundering period of cell, in reparation period after the damage that trysinization causes when being cell owing to inoculation, after this cell is exponential growth, i.e. exponential phase of growth, enter lag phase at last, the cell poor growth almost stops, and as seen has cell floating.And cell division index (MI) maximum (cultivating the 2nd day) occurs before entering logarithmic phase, and is maintained at a higher level when cultivating the 2nd～4 day, drops to initial level subsequently.The purity of the 'Jingning ' chicken embryo fibroblast series that the inventive method is cultivated is more than 99%, significantly improves than having with prior art culturing cell average purity 85%～90%; And population doubling time is 24h, compares with 48h with cell colony doubling time (PDT) 35.9h that prior art collagenase digesting technology and existing adherent culture method obtain to have significant raising, proves that cell purity height, vitality are vigorous.

Four, the cell motility rate is measured

Method:

The survival rate that detects freeze-stored cell can adopt the trypan blue exclusion test, will make cell suspension behind the cell recovery to be checked, gets 10 μ l cell suspensions and adds people 10 μ l 1% trypan blue dye liquor, mixing.Blood counting chamber counting, healthy viable cell cell space is complete, and cell is transparent, and is not painted, allly paintedly is blue person and is dead cell.Count 1000 cells, calculate cell survival rate.Several two kinds of total cellular score, the per-cent that accounts for total cellular score with viable cell reflects cell viability.

Conclusion:

Before and after the cell cryopreservation Jingning chick-embryo cell being carried out the cell motility rate measures.The result shows: the cell motility rate is between 95.8%～98.7% before frozen, and frozen back cell motility rate drops between 93.2%～96.7%, compares with prior art culturing cell motility rate about 80%～85% to increase significantly.And the recovery cell with frozen before compare, cell type still is an inoblast, the cellular form and the speed of growth do not change, the freeze-stored cell steady quality.

Five, the preparation of caryogram

Method:

1, add colchicine: the cell culture in the vegetative period of taking the logarithm, add colchicine in nutrient solution, final concentration is 0.1～0.4 μ g/ml.

2, in 37 ℃ of incubators, continue to cultivate 1～4h, make most of cell be in metaphase.

3, gather division phase cell: add 0.25% trypsin solution and digest, add full nutrient solution then, stop the effect of trypsin solution pair cell with ordinary method.Change the 15ml centrifuge tube over to, with the centrifugal 8min of 1000rpm, collecting cell.

4, hypotonic: absorb supernatant liquor, adding is preheated to 37 ℃, 0.075M (or 0.4%) KCl solution 2ml, beats and spares, and continues to add to 10ml, incubation 15min in 37 ℃ of incubators.

5, pre-fix: the fresh stationary liquid of adding in the suspension (acetic acid: 1ml methyl alcohol=1: 3), beat even (beat will blow and beat gently when sparing and prevent cell rupture).

6, fixing: suspension with the centrifugal 8min of 1000rpm, is removed supernatant, add fresh stationary liquid 5ml, beat and spare, room temperature leaves standstill 20min.

7, heavily fixing: as to repeat 2 times, remove the part supernatant after centrifugal, residue 1～1.5ml, mixing.

8, drip sheet: with slide glass inclination at 45, get rid of after the frozen water and drip last 2～3 cell suspensions rapidly with suction pipe, blowing on slice, thin piece with mouth immediately is uniformly dispersed cell, and slide glass is skimmed over spirit lamp several times, to help karyomit(e) dispersion and expansion, drying under the room temperature.

9, dyeing: with the Giemsa liquid dyeing 10min of 10 times of phosphate buffered saline buffer (pH6.8) dilutions, tap water flushing, dry air.

10, microscopy: find out the scattered division phase visual field with low power objective earlier, change high power objective and oily sem observation again, should see a lot of metacinesis phase cells and the karyomit(e) of sprawling, tenuigenin is dyed blueness, and nucleus is dyed lavender.

11, karyomit(e) photo and data processing: photomicrography is carried out in selective staining volume morphing and finely disseminated division mutually, 100 divisions of each kind statistics are to determine chromosome number, measure and calculate chromosomal three parameters promptly by Denver (1960) meeting and Leven standard (1964): relative length, arm ratio and centromere index, and definite kinetochore type.Three CALCULATION OF PARAMETERS formula are as follows:

12, need preserve or do long-time observation as sample, can cross twice of dimethylbenzene after, with neutral gum cover glass mounting, (or the earlier rapid acetone twice of crossing, each 30s, after dimethylbenzene, then mounting also can).

Conclusion:

The international standard of clone quality is that the occurrence rate of diploid cell reaches more than 85%, in this test, to the chromosome number counting of 100 Jingning chick embryo fibroblast, whether be that whole diploid is calculated only to it, the cell with the 1st～3 generation is a research object respectively.Statistics is: diploid accounts for main body in the cell chromosome, is more than 95%, illustrates that the 'Jingning ' chicken embryo fibroblast series of building reaches the standard of high-quality cell, the cytogenetics stable performance, and this cell is to stablize diploid cell line.

Concrete data see Table 2, and M represents metacentric chromosome, and SM represents submetacentric chromosome, and ST represents kinetochore, inferior end karyomit(e), and T represents kinetochore, end karyomit(e).

Jingning chicken diploid chromosome number order be 2n=78 ±, comprise 9 pairs of macrochromosomes and 30 pairs of minute chromosomes, sex chromosome is Z, W, and is male for joining ZZ, femalely is the different ZW that joins.As shown in Figure 8, as follows according to every pair of feature of karyomit(e) size order:

No. 1: submetacentric chromosome, a pair of for maximum;

No. 2: submetacentric chromosome is only second to number one in size;

No. 3: submetacentric chromosome;

No. 4: telochromosome, size is similar with No. three karyomit(e);

No. 5: the median kinetochore Z chromosome, another is inferior median kinetochore W karyomit(e);

No. 6: submetacentric chromosome;

No. 7: submetacentric chromosome;

No. 8: telochromosome;

No. 9: telochromosome;

10～No. 39: near-end or telochromosome;

Table 2 Jingning chicken karyomit(e) parameter (♀)

Six, isozyme separates

Method: adopt the discontinuous gradient polyacrylamide gel electrophoresis.

1, collecting cell: suspend numeration with PBS again;

2, clean: wash cell 3 times with PBS, centrifugal supernatant discarded night;

3, with protein extract suspension cell again, density reaches 5 * 10 ⁷Individual/ml, piping and druming evenly gently;

4, cell suspension is moved on in the 1.5ml centrifuge tube, collect suspension with the centrifugal 2min of 8.733g under 4 ℃, it is standby to go up suspension after the packing and put-70 ℃ of storages;

5, go up suspension and add equivalent 40% sucrose solution, mixing is sample liquid;

6, use micropipette to each sample cell application of sample 20～50 μ l, the electrode buffer with 10 times of dilutions is additional full each sample cell again, and groove slowly adds electrode buffer up and down then.In last groove, add 1～2 tetrabromophenol sulfonphthalein, put into 4 ℃ of refrigerators;

7, connect power supply, last groove is a negative pole, and following groove is anodal, and regulating voltage is that 120V keeps 1h, transfers to 220V again, treats that tetrabromophenol sulfonphthalein migrates to 0.5～1cm place, lower end and stops electrophoresis, about 5h;

8, electrophoresis finishes, the groove damping fluid is filled water with the 10ml syringe about collecting respectively, loads onto the long thin piercing needle of 10cm, inserts along glass inner side, advance in the marginal not waterside, the effect of cutting of scraping by the lubrication of water and syringe needle makes glue separate with sheet glass, picks up upper glass plate then gently, cut concentrated glue, after using twice of distilled water rinsing again, immersing insulation dyeing 30～60min in the mid-37 ℃ of incubators of staining fluid, takes a picture with the fixing back of gel stationary liquid in adhesive tape dyeing back;

9, with relative mobility (Rf) expression, promptly band swimming in district's is apart from (d) ratio with indicating dye swimming distance (D), and calculation formula is Rf=d/D * 100%.The range observation that district's band swimming distance is pressed district's band trailing edge without exception.

Conclusion:

As shown in Figure 9, the lactic dehydrogenase isozyme zymogram of Jingning chick embryo fibroblast is respectively LDH1, LDH2 from " anode " to " negative electrode ", LDH3, LDH4, LDH5, the enzymic activity power is followed successively by LDH5＞LDH4＞LDH3＞LDH2＞LDH1, and the LDH1 activity is very weak.Compare with the lactic dehydrogenase isozyme of white eared pheasant, what the result showed two chicken kinds has the feature pedigree separately, and relative mobility is different fully, sees Table 3.

The relative mobility of table 3LDH isozyme

As shown in figure 10, the malate dehydrogenase zymogram of Jingning chicken respectively presents 2 district's bands, and promptly sMDH (cytoplasm type) district's band group and mMDH (plastosome type) distinguish the band group, and the latter is slower than the former mobility speed.The relative mobility of two band groups sees Table 4.

The relative mobility of table 4 malate dehydrogenase (malic acid dehydrogenase)

The same enzyme is the different bands of a spectrum of performance in different species, in the same species different tissues.Above-mentioned two kinds of isozyme electrophoresis results show that the somatocyte heritability of being cultivated is stable, and crossed contamination does not take place between the cell purity height.

Seven, the expression of foreign gene in culturing cell

Method:

1, the preparation and the evaluation of fluorescin reporter plasmid (pEGFP-N3, pDsRed1-N1, pEYFP-N1): extract the box specification sheets according to Plasmid Midi Kit plasmid, extraction, purifying pEGFP-N3, pDsRed1-N1, pEYFP-N1 fluorescence protein gene plasmid.The gained plasmid is dissolved in TE (PH8.0) the less salt lysate, measure its plasmid concentration (plasmid concentration [μ g/ml]=OD260 * 50 μ g/ml * extension rates) with ultraviolet spectrophotometer, the pEGFP-N3 that obtains, pDsRed1-N1, pEYFP-N1 fluorescin plasmid concentration are 0.31 μ g/ml plasmid DNA, the OD260/OD280 value is between 1.80～1.86, experimental result show the plasmid that extracts purer, impurity such as no RNA, dehydrated alcohol and protein.After using Nhe I and Xba I double digestion again, cut the result, can obtain two bands of a spectrum of about 750bp and 4kb with 1% agarose electrophoresis evaluation enzyme.Really be required pEGFP-N3, pDsRed1-N1, pEYFP-N1 fluorescin plasmid according to fluorescin plasmid enzyme restriction plasmid that atlas analysis obtains.

2, liposome mediated-method transfection inoblast

(1), transfection the day before yesterday, with trypsin solution peptic cell culture, seed cells in the culture plate of 6 holes (or 35mm) every hole (1～2) * 10 ⁵Cell adds 2ml and contains the serum nutrient solution;

(2), put 37 ℃ CO ₂Culturing cell reaches 70%～80% the degree of converging in the incubator;

(3), before the transfection, under inverted microscope, observe, picking growth cell vigorous, that form is good is used for transfection;

(4), will treat that cells transfected does not contain twice in the nutrient solution rinsing cell of serum;

(5), merge solution A and B, mix the back gently and under room temperature, leave standstill 45min, add the nutrient solution that 0.8ml does not contain serum then;

(6), DNA, liposome and the mixture that do not contain the nutrient solution of serum dripped in cultivating version treat the cells transfected surface, in CO ₂Continue to cultivate 5～24h in the incubator;

(7), outwell transfection night, add the nutrient solution that contains serum, in incubator, continue culturing cell;

(8), the expression of results of under the respective color fluorescence excitation, observing three kinds of fluorescins, calculate transfection efficiency.

Conclusion:

Shown in Figure 11,12, after the transfection peak of Jingning chick embryo fibroblast appeared at transfection 24h, the fluorescence intensity of green fluorescent protein reached changeing that 48h is the strongest then, and transfection efficiency is 55.5%; Red fluorescent protein pDsRed1-N1 48h transfection efficiency after transfection reaches 60.2%, as shown in figure 13; Yellow fluorescence protein pEYFP-N1 48h transfection efficiency after transfection has also reached 60.2% respectively, as shown in figure 14; Positive cell and fluorescence intensity all reduce gradually, weaken behind the transfection 48h.

The method that foreign gene is changed over to cell is a lot, mainly contains liposome, poly-lysine mediation, acceptor Jie high-voltage electric shock, particle gun and microinjection etc.Lipid mediation transfection is the most effective at present transfection method, and the used DNA amount of lipid mediation transfection is compared with calcium phosphate method greatly and reduced, and transfection efficiency exceeds 5～100 times than calcium phosphate method.By liposome-mediated plasmid DNA to the inoblast of Jingning chicken the transfection of sampling, expression effect by the observation fluorescence protein gene oppositely confirms the heritability of institute's culturing cell, and the result shows that the transfection efficiency of green fluorescent protein pEGFP-N3 is 55.5%; The transfection efficiency of yellow fluorescence protein pEYFP-N1 has also reached 60.2% respectively, as shown in figure 14; The transfection efficiency of red fluorescent protein pDsRed1-N1 reaches 60.2%, is higher than 15%～30% transfection efficiency of common culturing cell far away.These presentation of results the Jingning chick fibroblast of being cultivated expression alien gene well, further proved the characteristic of the high expression level rate of culturing cell system of institute.

Eight, apoptosis

Method:

1, cell is inoculated in 12 well culture plates with 4.0 * 105/ml density, is cultured to cell attachment among the every hole 3ml, 37 ℃, 5%CO2 incubator.

2, DAPI dyeing: outwell the substratum of cell, cover cell with DAPI-methyl alcohol working fluid again after the DAPI-methyl alcohol working fluid flushing once with 1 μ g/ml, 37 ℃ of incubation 15min outwell staining fluid, with washed with methanol once again.

3. observe under the laser scanning co-focusing microscope and take pictures.

Conclusion:

Apoptosis is a life and death alternative natural process of finishing new and old cell, if the not normal then individuality of its rule promptly can not normal development, or deformity takes place, and maybe can not survive.

During apoptosis, nuclear chromatin is condensed into piece and condenses upon around the nuclear membrane, and after birth caves in cell is divided into a plurality of have adventitia parcel, the not excessive apoptotic bodies of inclusion voluntarily.Its reason is because eukaryotic cell chromatin is to be packed in some way by many nucleosomes to form, nucleosome is made up of core particle and joining region two portions, the former is made up of the dna fragmentation of octameric histone and 140bp, the latter is the starting point of synthetic RNA primer, form by the dna fragmentation about histone h1 and 60bp, H1 phosphorylation and cell fission are in close relations, are subject to endonuclease and attack.Each nucleosome DNA total length is 150～241bp.During apoptosis, activated endonuclease, cutting joining region DNA, thus generation length is the oligonucleotide fragment about 180～200bp, forms apoptotic body.

The specificity fluorescent dyestuff DAPI of DNA therefore commonly used combines with the A-T base district of DNA with non-embedded, thus the pair cell nuclear staining.After the dyeing, viable cell nuclear is the uniform blue-fluorescence of disperse under the laser scanning co-focusing microscope, when apoptosis occurring, and the visible dense block fluorescence of fine and close blue particle, apoptotic cell when Figure 20 is Jingning chicken recovery of dying in nucleus or the tenuigenin.

Under the Laser Scanning Confocal Microscope, observe 100～200 cells of statistics, calculate apoptosis rate:

Apoptosis rate=apoptosis cell/total cell count * 100%

Calculate the 'Jingning ' chicken embryo fibroblast series apoptosis rate of being cultivated and be lower than 5%.

Embodiment 3 utilizes the Jingning chicken to be made into fibrocyte to carry out cytogamy experiment

1, cell dyeing

(1) Jingning chicken Yihong dyeing: 95% (volume ratio) ethanol 5s, Yihong staining fluid dyeing 30s～120s (can and require the adjustment time) according to coloration result.Get final product direct viewing after 70% (volume ratio) washing with alcohol 2 times.

(2) White Yak in Tianzhu DAPI dyeing: outwell the substratum of cell, cover cell with DAPI-methyl alcohol working fluid again after the DAPI-methyl alcohol working fluid flushing once with 1 μ g/ml, 37 ℃ of incubation 15min outwell staining fluid, with washed with methanol once again.

2, cell mixing

Get 10 of 4ml recovery back cultivation ⁶～10 ⁷Centrifuge tube at the bottom of individual/ml cell suspension injection 10ml tool plug tip, the suspension of taking-up 0.4ml is with 5 times of physiological saline dilutions, group in contrast.

3, cytogamy

(1) with above-mentioned remaining cell suspension with the centrifugal 5min of the rotating speed of 1500r/min, remove most of supernatant liquor, stay 0.2ml liquid settled cell be scattered in wherein, make cell suspension;

(2) shake test tube gently, and dropwise add 50% (mass volume ratio) PEG solution 0.4ml of 37 ℃ of following preheatings;

(3) this suspension is placed 37 ℃ of water-baths, incubation 90s;

(4) add serum-free medium 5ml lentamente, to end the effect of PEG;

(5) capping plug, and the centrifuge tube 4～5 times of verting slowly;

(6) with the centrifugal 5min of 1500r/min.Remove most of supernatant liquor, stay about 0.1ml liquid suspension cell;

(7) the full nutrient solution of adding 5ml in suspension behind the mixing, takes out the 0.4ml suspension and drips sheet do observation usefulness.

4, cell cultures

Cell is done 4 times of dilutions, and mixing is sub-packed in 24 well culture plates or every hole 0.1ml is sub-packed in 96 well culture plates with every hole 0.5ml, places 37 ℃, 5%CO ₂CO ₂Cultivate in the incubator.

5, observe with laser scanning co-focusing microscope

From two kinds of parental cells of form identification and observe and wherein have or not fused cell, simultaneously by laser scanning co-focusing microscope observe same cell caryoplasm color different be fused cell, as Figure 15.

6, cell confluency

Observe statistics 100～200 cells (comprise merged with do not merge), calculate fusion rate:

Fusion rate=fused cell number/total cell count * 100%

Conclusion:

Though the PEG fusion method does not have specificity or specificity between kind kind section, the restriction between animals and plants is also broken, and its fusion rate is low.Under the top condition, the fusion rate between not equal genus protoplastis only is 30%, and PEG method pair cell toxicity is big in addition.Therefore, the fusion of PEG inducing cell selects relative molecular mass 1000～6000 usually, and concentration is at the PEG of volume ratio 30%～50% (mass volume ratio), and strict control urgees to melt action time the toxicity of minimizing pair cell.

White Yak in Tianzhu nucleus and Jingning chicken cell matter is respectively with after the dyeing of DAPI and Yihong, again with relative molecular mass be 4000, to be that the PEG of 50% (mass volume ratio) is short melt 90s to concentration, thereby make nuclear and matter dye the cytogamy of indigo plant, redness respectively.Allos cell or protoplastis are under the fusogen effect, the cytolemma character that is fine and close state cell changes, iuntercellular generation agglutination phenomenon, and the intercellular film of a part of aggegation sticks together, be fused into syncyte, the fusion examined again of syncyte subsequently and become the monokaryon hybrid cell.

Figure 15 is for being presented red Jingning chicken cell matter by Yihong is painted under the 543nm wavelength exciting light, and Figure 16 is for being blue White Yak in Tianzhu nucleus by fluorescence dye DAPI is painted under the 408nm wavelength excitation.Because a kind of nuclei dyeing is blue, another kind of tenuigenin dyes and is redness, so contain the cell that is fusion of blue nuclear and erythrophane in the same cell, the composite diagram of cell under two kinds of exciting light effects under the same visual field as shown in figure 17, Figure 18 is White Yak in Tianzhu and a certain site of chicken fused cell, Jingning enlarged view, and Figure 19 is that Jingning chicken cell self merges (400 *).

By calculating: the cell confluency of White Yak in Tianzhu and Jingning chicken is 97.2%, and Jingning chicken self cell confluency is 92.4%.Fusion rate is higher, this be because with merge before the dyeing processing carried out of pair cell, alcohols degreasing dehydration fixing, thereby changed membrane structure, improved fusion rate; Can find out also that simultaneously of the same race and not of the same race 's cell confluency is basic identical.

Above presentation of results the 'Jingning ' chicken embryo fibroblast series cultivated can be used for the cytologic experiment of wider scope such as cytogamy, for other biological is learned, medical field provides high-quality clone.

Claims

1, a kind of 'Jingning ' chicken embryo fibroblast series is characterized in that, its deposit number is CGMCC No.1878.

2, a kind of cultural method of 'Jingning ' chicken embryo fibroblast series is characterized in that, this method comprises the steps:

(1) first culture: will cut into 0.5～1.5mm with after the PBS rinsing 3～4 times except that the Jingning Embryo Gallus domesticus of decerebrate, eye, four limbs, internal organ ³Tissue block; Tissue block is moved into culturing bottle, be inverted culturing bottle, and at 37.5 ℃, 5%CO ₂CO ₂Cultivate 3～4h in the incubator; The complete adherent back of tissue block adds nutrient solution 6～10ml, the nutrient solution composition: the MEM+10% foetal calf serum, continue to cultivate 10～12h;

(2) cultivation of going down to posterity: the nutrient solution in reject step (1) culturing bottle behind residue in the PBS washing culturing bottle 2 times, stops digestion with the MEM 6～10ml that adds full nutrient solution 10%FBS behind the tryptic digestion; Postdigestive cell average mark is packed in 2 culturing bottles, puts into 37.5 ℃, 5%CO ₂CO ₂Continue to be cultured to frozen preceding 24h in the incubator;

(3) cell cryopreservation:

Centrifugal 6～the 10min of D, collection: 1000rpm removes supernatant liquor, adds the frozen storing liquid 1ml of 4 ℃ of precoolings, makes cell resuspended with suction pipe piping and druming behind the mixing; Frozen storing liquid composition: 10%DMSO+50% foetal calf serum+40%MEM;

E, the culture packing gone in the frozen pipe of sterilization to seal;

3, the cultural method of a kind of 'Jingning ' chicken embryo fibroblast series according to claim 2 is characterized in that, the Jingning Embryo Gallus domesticus described in the step (1) is 6 ages in days.

4, the cultural method of a kind of 'Jingning ' chicken embryo fibroblast series according to claim 2, it is characterized in that, trysinization concrete steps described in step (2) and the step (3) are: add 0.25% trypsinase, 1.5～2.5ml in culturing bottle, be inverted the digestion 30s～60s that overturns culturing bottle is preheated to 37 ℃ in incubator after.

5, the cultural method of a kind of 'Jingning ' chicken embryo fibroblast series according to claim 2, it is characterized in that, the cell that step (3) is frozen carries out cell recovery, concrete steps are: frozen pipe is taken out from liquid nitrogen insert in 42 ℃ of water-baths, after rocking 1 fen kind continuously, cell moved in the culturing bottle be added with nutrient solution MEM+10% foetal calf serum piping and druming evenly, place and contain 37.5 ℃ of 5%CO ₂CO ₂Can continue the cultivation of going down to posterity after continuing in the incubator to cultivate 10～12h.