CN101020897A

CN101020897A - Ear margin tissue fibroblast line of Minxian county black fur sheep and its culture process

Info

Publication number: CN101020897A
Application number: CNA2006101619476A
Authority: CN
Inventors: 马月辉; 关伟军; 张艳艳; 马忠仁; 陈莉娜; 刘长青
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: 2007-08-22
Anticipated expiration: 2026-12-11
Also published as: CN100506976C

Abstract

The present invention is high activity and high purity fibroblast line of Minxian county black fur sheep obtained with the ear margin tissue and through primary cell culture, subculture and cell freezing preservation and in the preservation number of CGMCC No.1880, and belongs to the field of cell biology. The high activity and high purity fibroblast line is suitable for great scale culture. The present invention makes it possible to provide great amount of high quality fibroblast line of Minxian county black fur sheep for the scientific research in gene engineering, cell engineering, etc.

Description

Ear margin tissue fibroblast line of Minxian county black fur sheep and cultural method thereof

Technical field

This invention is about a kind of foundation of clone and cultured method thereof, promptly utilizes the ear-edge tissue of Minxian County black-fur sheep to carry out cellular segregation, first culture, going down to posterity to cultivate has obtained high motility rate, high purity ear margin tissue fibroblast line of Minxian county black fur sheep.The invention belongs to field of biology.

Background technology

The livestock and poultry genetic resources is the important component part of species diversity, also is that the same mankind concern the most closely, the most direct part, and in the whole world, the livestock and poultry genetic resources reaches more than 30% for the mankind provide food and agricultural-food.According to the prediction of government of developed country and the World Food Programme, agricultural 90% kind in the whole world all will provide and kind also will reach more than 50% the contribution rate that whole animal produces by molecular breeding in this century.Raising along with continuous change of working condition and living standards of the people, people increase the livestock product quality and quantity, requirement cultivate adaptability stronger, high yield and high quality, livestock and poultry species (being) with unique proterties, and these must rely on existing livestock and poultry genetic resources.Therefore strengthen to existing livestock and poultry genetic resources evaluation, preservation and effectively and reasonably, sustainable use is significant.

World Food and Argriculture OrganizationFAO (FAO) the animal genetic resources is defined as that institute's perch has changed on earth or still unaltered envrionment conditions under, the breeding of animal, kind, type and population quantity.Biological Genetic Resources is the important foundation of bio-science research, is human survival and social economy's STRATEGIES OF SUSTAINABLE DEVELOPMENT resource.In the world will be to the situation of occupying of Biological Genetic Resources as one of important indicator of weighing a national national power.The livestock and poultry genetic resources is important Biological resources, is human foods and healthy direct sources, is the essential starting material of genetically engineered and industrialized development.In most developed countries, along with the intensification of livestock production system, the just high kind and the cross-fertilize seed of minority economic worth of mass rearing, the kind number is in rapid minimizing; At some developing china familys,,, the quantity of original local variety is significantly reduced owing to protect kind of an improper and blind import adventive hybridization though more rich genetic resources is arranged.Thereby cause global genetic resources crisis.

Therefore, the protection of livestock and poultry genetic resources is the significant problem that is related to aquaculture sustainable development and species diversity.Protection livestock and poultry genetic resources is to the sustainable development of China and even world's livestock industry, and the development and use of Animal resources have great importance.Therefore, the importance of genetic resources is more and more recognized in countries in the world in recent years.In June, 1992; more than 150 country that comprises China signed Convention on biological Diversity jointly; its 3 major objectives are: the protection species diversity; the utilization of commercial benefits and other form of genetic resources is shared in the sustainable use of species diversity moiety in fair and reasonable mode.After this Convention on Biological Diversity has been passed through " Bonn criterion " and " Kuala Lumpur minister's declaration " again successively, the Biological Genetic Resources in the whole world is protected and managed.

According to nearest statistics; in 6165 livestock and poultry species in the whole world; have that 740 (accounting for 12%) are individually become extinct, 531 endangered (accounting for 8.16%), 1092 be in (accounting for 17.71%) in imminent danger, situation is unclear 1407 (accounting for 22.8%), the having only of non-crisis 2395 (accounting for 38.8%).The ratio that became extinct in 1999, kind endangered and in imminent danger accounts for the kind sum than nineteen ninety-five proportion increase by 82.09%, 37.54%, 19.02% respectively, worldwide livestock and poultry germ plasm resource criticality increasingly sharpens.Abroad, some government departments, organizational structure quite pay attention to the preservation and the management of livestock and poultry genetic resources as Food and Argriculture OrganizationFAO (FAO).

China is one of country that livestock and poultry local variety resource is the abundantest in the world.The excellent germplasm characteristic of local variety is the selected results of diversified for thousands of years natural ecological environment.Many good local livestock and poultry species have that adaptability is strong, crude feed tolerance, breeding potential height and product fine characteristics.By correlation study and audit, China's livestock and poultry genetic resources has 576 kinds (monoid), and wherein local variety (monoid) are 426, account for 74% of variety source sum; Cultivate 73 of kinds, account for 12.7% of variety source sum; 77 of introduced varieties account for 13.3% of variety source sum.These genetic resources characteristics are different, but owing to be subjected to influence of various factors such as a large amount of external high-yield variety introductions, some local variety is replaced by cross-fertilize seed gradually, have and enrich genetic local variety because constantly modified, quantity sharply reduces even withers away, and this trend is along with the raising further aggravation of the intensive degree of livestock and poultry.There are 158 to lose original breeding potentiality to a great extent in 576 main poultry kinds, account for 36.6% of China's poultry kind sum, wherein have 32 can not save or extinction because scale reduces.At beginning of the eighties late 1970s, in accounting for 77% local variety of kind sum, livestock and poultry species resource investigation result confirms that the kind that China has become extinct has 10,8 of endangered kinds, and what quantity reduced has 20.331 local livestock and poultry species multidate information resource explorations were economized in 17 in the whole nation in 1996～1998 years and shown that have 50 livestock and poultry species (or monoid) in imminent danger, 9 kinds (or monoid) are endangered, 7 kinds (or monoid) are become extinct.This trend is along with introducing a fine variety in a large number in recent years and the raising of the degree that intensifies and further aggravation, estimates to have at least 30% livestock and poultry genetic resources to be among the highly dangerous of extinction.

Therefore, 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 clone is preserved its genetic resources, and is significant.

The Minxian County black-fur sheep is the local variety of China's Gansu Province's fur coat with sheep, belongs to mountain valley-type and hides sheep, and be the local variety resource of a preciousness in China's sheep husbandry.Because the Minxian County black-fur sheep produced the hair fur coat have that heat absorption is warming, the hair fringe attractive in appearance, pitch-black smooth, wear long durable, do not lose hair or feathers, soft characteristics such as fine and closely woven of felt knot, dermatotome thereby welcome by consumers in general with the fur clothing of its processing and fabricating.The Minxian County black-fur sheep is put into 2006 " the national livestock and poultry genetic resources protection catalogue " announced, and is significant to the preservation of the genetic resources of Minxian County black-fur sheep.

Summary of the invention

The purpose of this invention is to provide high motility rate, highly purified ear margin tissue fibroblast line of Minxian county black fur sheep, its preserving number is: CGMCC No.1880.

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:

High motility rate, highly purified ear margin tissue fibroblast line of Minxian county black fur sheep are preserved (CGMCC, address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City by China Committee for Culture Collection of Microorganisms common micro-organisms center; Postcode: 100080; Phone: 010-62542758), preservation day: on December 1st, 2006, preserving number: CGMCCNo.1880, the classification called after ear margin tissue fibroblast line of Minxian county black fur sheep of suggestion.This ear margin tissue fibroblast line of Minxian county black fur sheep has following feature:

Promptly have cell to grow behind the Minxian County black-fur sheep tissue block inoculation 6h, 1～2d can stick that bottle wall visible cell under inverted microscope is transparent, and the kytoplasm projection is plentiful, is spindle shape, karyon ellipse, placed in the middle, and kernel is clear, and stereoscopic sensation is strong.When cell growth in flakes the back cellular form be that typical one-tenth is fibrous, be fusiformis or sealene triangle, that cell is when growing is radial, flamboyancy or swirl shape are out of shape.High cell growth speed, surviving rate height, exogenous gene expression rate height, applied range.

The cultural method of a kind of high motility rate, high purity ear rim tissue fibroblast series, this method comprises the steps:

(1) first culture: the Minxian County black-fur sheep ear-edge tissue that will adopt cuts into 0.5～1.5mm with adding after 1% couple of anti-PBS cleans 6～8 times ³Tissue block; At the bottom of tissue block moved into culturing bottle and being tiled in bottle, be inverted and be placed on 37 ℃, 5%CO ₂Incubator in cultivate 3～4h; Treat that the complete adherent back of tissue block adds substratum 8～10ml, the superfine foetal calf serum (FBS) of nutrient solution composition: DMEM+10%.

(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 adding nutrient solution 6～10ml behind the tryptic digestion; Postdigestive cell suspension average mark is packed in 2 culturing bottles, puts into 37 ℃, 5%CO ₂Incubator in continue to cultivate.

(3) cell cryopreservation:

A, change liquid: nutrient solution is also changed fresh medium 6～10ml in the 24h before frozen, reject culturing bottle, continues to cultivate 24h;

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

C, counting: calculate frozen preceding total cellular score with the red blood cell count(RBC) plate;

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%DMEM;

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

F, pre-freeze: with frozen pipe load program the cooling box in, place 4 ℃ of 20～30min, change-70 ℃ of pre-freeze 4～6h then over to;

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

In the cultural method method of above-mentioned high motility rate, high purity ear margin tissue fibroblast line of Minxian county black fur sheep, the Minxian County black-fur sheep material that step (1) is got is the best with the ear-edge tissue piece.

The cultural method of above-mentioned high motility rate, high purity ear margin tissue fibroblast line of Minxian county black fur sheep, wherein, the concrete steps of trypsin digestion are described in step (2) and the step (3): add 0.25% trypsinase, 1～2ml in culturing bottle, the inversion culturing bottle is preheated to 37 ℃ in incubator after, upset digestion 30～60s.

The cultural method of above-mentioned high motility rate, high purity ear margin tissue fibroblast line of Minxian county black fur sheep, wherein, the frozen cell of step (3) can be recovered according to actual needs, concrete steps are: frozen pipe is taken out from liquid nitrogen put into 42 ℃ of water-baths rapidly, and ceaselessly rock it is evenly thawed, after thawing fully, cell moved in the culturing bottle be added with nutrient solution piping and druming evenly, the centrifugal 8min of 1000rpm is with flush away DMSO, cell after centrifugal uses nutrient solution resuspended again, places to contain 37 ℃ 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 adjusting and improving adherent culture method and nutrient solution composition, can make the inoblast of turning out not have impurity cells such as epithelial cell, and cell purity has significantly raising than prior art; The cell that the improvement of frozen condition is made recovery 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 cell motility rate behind the cell cryopreservation can reach 92.4%～98.6%, and the passage growth is stable, compares with existing culture technique cultured cells to have a clear superiority in, and is fit to large scale culturing.More than to the adjustment of nutrient solution and the improvement of cultural method, significantly reduced cost.In addition, the present invention has also remedied the deficiency that existing carcass cell is preserved in each side such as methods, and the lifting of ear margin tissue fibroblast line of Minxian county black fur sheep motility rate and the purity genetic resources that also makes Minxian County important, in imminent danger black-fur sheep kind is able to prolonged preservation with the form of cultured cell in vitro.

High motility rate of the present invention and highly purified ear margin tissue fibroblast line of Minxian county black fur sheep 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 somatic cell clone breeding; On agricultural, can enrich, improve local variety; Can also be as the main raw material(s) of production of vaccine and the feeder layer of stem cell cultivation; 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 Minxian County black-fur sheep ear rim tissue fibroblast microcytoscope figure below;

Fig. 2 is Minxian County black-fur sheep ear rim tissue fibroblast cell subculture I microcytoscope figure below;

Fig. 3 is Minxian County black-fur sheep ear rim tissue fibroblast cell subculture II microcytoscope figure below;

Fig. 4 is by the inoblast of mycoplasma contamination contrast figure;

Fig. 5 is a Minxian County black-fur sheep ear rim tissue fibroblast cell detection of mycoplasma negative findings diagram;

Fig. 6 is Minxian County black-fur sheep ear rim tissue fibroblast cell growth curve figure;

Fig. 7 is a Minxian County black-fur sheep ear rim tissue fibroblast cell caryogram diagram;

Fig. 8 is a Minxian County black-fur sheep ear rim tissue fibroblast cell lactic dehydrogenase isozyme electrophoresis diagram;

Fig. 9 is a Minxian County black-fur sheep ear rim tissue fibroblast cell malate dehydrogenase (malic acid dehydrogenase) electrophoresis diagram;

Figure 10 is that the pEYFP-N1 gene is expressed 100 times of diagrams of 48h in the black-fur sheep ear rim tissue fibroblast cell of Minxian County;

Figure 11 is that gene is expressed 100 times of diagrams of 72h in the black-fur sheep ear rim tissue fibroblast cell of Minxian County;

Figure 12 is 400 times of diagrams of pEYFP-N1 fluorescin plasmid transfection cell strain;

Embodiment

Used key instrument and reagent source among the embodiment:

Black-fur sheep source, Minxian County: Minxian County, Gansu Province

(1) key instrument equipment

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

2, embossment phase microscope, Olympus IX-71;

3, biomicroscope, Olympus CX31;

4, inversion differs fluorescent microscope: Olympus IX-71;

5 ,-70 ℃ Ultralow Temperature Freezer: U.S. kelvinator;

6, electronic pipettor: German Accu-jet;

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

8, CO2 incubator: Heraeus BB16UV;

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

10, electrophoresis apparatus: Beijing Liu Yichang DYY-6C;

11, electrophoresis chamber: Beijing Liu Yichang DYC-24A:

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

13, low speed centrifuge: CENTERIGUGETDL-40B;

(2) main agents

1、DMEM(Dulbecco’s modified eagle medium)；

2, Plasmid Midi Kit plasmid extracts box, Trizol reagent, cDNA synthetic agent box, cDNA PCR library test kit, G418, pEGEP-N3 plasmid vector: Clontech;

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

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

5, trypan blue (Trypan Blue), DMSO, colchicine (Colchicine), EDTA, Triton (Triton) X-100＞99%, TEMED, Bis (methylene diacrylamide) 98%, ammonium persulphate (Ammonium Persulfate), PMS, NAD, tetrazolium bromide (Thiazolyl Blue), cytochalasin B, Hoechst33342, ionomycin, 6-DMAP:Sigma;

6, glycerine analytical pure Hua Lvyuan;

7、Tris：Promega；

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

9, liposome Lipofectin:Gibco company;

Inoblast is cultivated used liquid dosage:

10, DMEM nutrient solution: DMEM 9.6g, be dissolved in the ultrapure water, use NaHCO ₃About 3.2g regulates pH to 7.2～7.4, adds water and is settled to 1L, uses the magnetic stirrer mixing, 0.22 μ m membrane filtration degerming, and the packing of 500ml/ bottle is stored in 4 ℃.

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

12, 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.

13,1 * PBS damping fluid: NaCl 4.00g, KCl 0.10g, Na ₂HPO ₄12H ₂O 1.45g, KH ₂PO ₄0.10g, adding ultrapure water and be settled to 500ml, pH is 7.2, autoclaving seals back 4 ℃ of storages.

14,0.25% (mass volume ratio) trypsin solution: 1.25g trypsinase dry powder is dissolved among the PBS, is settled to 500ml, and pH is 7.2～7.4,0.22 μ m membrane filtration packing ,-20 ℃ of storages.

15,0.9% (mass ratio) physiological saline: 0.45g NaCl is dissolved in the 500ml ultrapure water, autoclaving 30min.

16, 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 0.22 μ m membrane filtration 100ml/ bottle packing.The microorganism detection agents useful for same:

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

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

19, DAPI dye liquor: ultrapure water preparation 1mg/ml DAPI storage liquid, the PBS dilution is 0.5～1mg/ml.

20, 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 ℃.

21, 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:

22, colchicine stock solution: the 10mg colchicine is dissolved in the 10ml ultrapure water.

23, diluent: ultrapure water is 100 μ g/ml with 10 times of stock solution dilutions to final concentration.

24, hypotonic KCl solution: the KCl of 0.5g is dissolved in the 100ml ultrapure water.

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

26, 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.

27, the preparation of phosphoric acid buffer: KH ₂PO ₄0.34g add 100ml deionized water (transferring pH 6.8) liposome transfection cell agents useful for same with NaOH:

28, plasmid DNA (EGFP-N3), liposome Lipofectamine does not contain the DMEM nutrient solution of serum, contains the DMEM nutrient solution of serum, the PBS of pH 7.4.

The Isozyme Analysis agents useful for same:

29,0.9% (mass ratio) NaCl-0.06mmol EDTA solution: take by weighing 0.9g NaCl and 0.0223g EDTA respectively and be dissolved in the 100ml ultrapure water and get final product.

30, 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-0.06mmol EDTA solution.TritonX-100: 0.9% (mass ratio) NaCl-0.06mmol EDTA solution=1: 15.

31,40% (mass volume ratio) sucrose solution: 40g sucrose is dissolved in the 100ml water.

32, 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

33, polyacrylamide gelating soln concentration sees Table 1

Table 1

The enzyme class	Separation gel			Concentrate glue			Electrode buffer
	Separation gel			Concentrate glue			Electrode buffer	Concentration	Degree of crosslinking	pH	Concentration	Degree of crosslinking	pH	pH
	LDH	7.5	2.6	8.9	3	2.1	6.7	Concentration	Degree of crosslinking	pH	Concentration	Degree of crosslinking	pH	pH	8.3
MDH	LDH	7.5	2.6	8.9	3	2.1	6.7	8	2.5	8.9	3	2.1	6.7	8.3	8.3

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

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

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

Calcium lactate 100mg+0.05M Tris-HCl (pH8.0) 20ml

Tetrazolium bromide 5mg+ ultrapure water 1.0ml

PMS 2.5mg+ ultrapure 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.

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

DL-oxysuccinic acid 350mg is dissolved among the 25ml 0.1M Tris-HCl (pH8.0)

Tetrazolium bromide 15mg+ ultrapure water 1.5ml

PMS 5mg+ ultrapure water 1.0ml

Nadide (NAD) 10mg

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

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

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

40, stationary liquid

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

41, DAPI dye liquor: ultrapure water preparation 1mg/ml DAPI storage liquid, the PBS dilution is 0.5～1mg/ml.

42, 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 structure of embodiment 1 high cell motility rate high purity ear margin tissue fibroblast line of Minxian county black fur sheep

(1) first culture: under the aseptic condition, Minxian County black-fur sheep ear-edge tissue sample is scraped off the hair on surface with eye scissors, put into the culture dish rinsing 6～8 times that PBS is housed, to remove surperficial blood stains and hair; Tissue is cut into 1mm ³About tissue 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%CO ₂CO ₂Cultivate 2～3h in the incubator; The complete adherent back of tissue block adds substratum 8ml～10ml, and the superfine foetal calf serum of medium component: DMEM+8% 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, 1～2ml, after being inverted the digestion 30～60s 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 (DMEM of 10%FBS) 15ml～20ml and stops digestion; Postdigestive cell average mark is packed in 2 culturing bottles, puts into 37 ℃, 5%CO ₂CO ₂Continue in the incubator to cultivate.

(3) cell cryopreservation:

A, frozen preceding 24h, nutrient solution is also changed fresh medium 8～10ml in the reject culturing bottle, and the superfine foetal calf serum of nutrient solution composition: DMEM+10% continues to cultivate 24h;

B, in culturing bottle, add 0.25% trypsinase, 1～2ml, behind upset digestion 30～60s, add full nutrient solution 8ml～10ml termination reaction after being inverted culturing bottle and in incubator, being preheated to 37 ℃;

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

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

F, pre-freeze: frozen pipe is loaded program in the cooling box, be put in 4 ℃ of 20～30min, then in-70 ℃ of refrigerators more than the pre-freeze 4h;

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

(4) cell recovery: frozen pipe is taken out from liquid nitrogen, inserts rapidly in 42 ℃ of water-baths, rock 1min continuously after, see the surplus Bechtop of putting into when having little ice group to become the soybean grain size; Cell moved in the culturing bottle be added with nutrient solution (the superfine foetal calf serum of DMEM+8%) piping and druming gently evenly, place and contain 37 ℃ of 5%CO ₂CO ₂Can continue the cultivation of going down to posterity after continuing in the incubator to cultivate 10～12h.

The detection of embodiment 2 ear margin tissue fibroblast line of Minxian county black fur sheep biological characteristicses

The ear margin tissue fibroblast line of Minxian county black fur sheep 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 (Minxian County black-fur sheep primary cell), Fig. 2 (Minxian County black-fur sheep go down to posterity preceding cell), Fig. 3 (Minxian County black-fur sheep subculture 1 cell), Fig. 4 (Minxian County black-fur sheep embryo 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 freeze-stored cell amount 0.5%, mixing in 8ml nonreactive nutrient solution, cell suspension be with the centrifugal 20min of 1000rpm, 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: the visual inspection inoculation has the soybean Tryptones nutrient solution and the wort nutrient solution of Minxian County black-fur sheep ear rim tissue fibroblast cell, 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)～operation of (4).

Conclusion:

Because 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 Minxian County black-fur sheep ear rim tissue fibroblast cell of cultivating under the condition of antibiotic-free, to observe at phase microscope, 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 2ml; Add sample 2ml to be checked in the ware to be checked, before cultured cells is converged on the cover plate, from bottle, take out (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 *～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 complete cytolemma, in the intercalation of 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 black-fur sheep ear rim tissue fibroblast cell film-making of vitro culture Minxian County, under inverted fluorescence microscope, purple fluorescence with 380nm excites, can find that the visible cell 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.Prove that this cell does not have mycoplasma contamination.

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 well culture plates, inoculate the cell of equal amts respectively, general 1～2 * 10 ⁴Individual/ml, in 37 ℃ of CO ₂Continue in the incubator to cultivate.

(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 Minxian County black-fur sheep 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 continuous 7d regularly to count, write down each time cell density, draw and form the culturing cell growth curve as shown in Figure 7, inoculation back 2d cell count begins to increase, 3d enters logarithmic phase, 6d 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 2d) occurs before entering logarithmic phase, and is maintained at a higher level when cultivating the 2nd～4d, drops to initial level subsequently.The purity 98.9% of the ear margin tissue fibroblast line of Minxian county black fur sheep that the inventive method is cultivated is compared with prior art culturing cell average purity 91% and is significantly increased; And the cell colony doubling time (PDT) is 24h, compares with 48h with 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 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.Add up two kinds of total cellular score, account for the per-cent reflection cell viability of total cellular score with viable cell.

Conclusion:

Before and after the cell cryopreservation Minxian County black-fur sheep ear-edge tissue 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 is 93.2%～96.7%, compares with the frozen back of existing cell culture technology cell alive 78.6% 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 nutrient solution then, stop the effect of trypsinase pair cell with ordinary method.Change the 15ml centrifuge tube over to, with the centrifugal 8min of 1000rpm, collecting cell.

4, hypotonic: sedimentation cell is with being preheated to 37 ℃, 0.075M KCl solution 2ml, resuspended, after the piping and druming evenly, continue to add KCl solution to 10ml, put into 37 ℃ of incubator incubation 15min.

5, pre-fix: the fresh stationary liquid of adding in the suspension (acetic acid: 1ml methyl alcohol=1: 3), beat even gently.

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: drip 2～3 cell suspensions in 45 ° of inclination slide glasss, cell is uniformly dispersed, dry 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: the visible metacinesis phase of oily sem observation cell and the karyomit(e) of sprawling, tenuigenin is dyed blueness, and nucleus is dyed lavender.

11, karyomit(e) photo and data processing: 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, mounting: after crossing twice of dimethylbenzene, with neutral gum cover glass mounting.

Conclusion:

One of international standard of clone quality is that the occurrence rate of diploid cell reaches more than 85%, in this test, chromosome number counting to 100 Minxian County black-fur sheep ear rim tissue fibroblast cells, whether be that whole diploid is calculated to it only, the cell with the 1st～3 generation is a research object respectively.Statistics is: diploid cell accounts for main body, is more than 95%, illustrates that the ear margin tissue fibroblast line of Minxian county black fur sheep of building reaches the standard of high-quality cell, the cytogenetics stable performance.

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).

Minxian County black-fur sheep diploid chromosome number order is 2n=54, comprises 26 pairs of euchromosomes and a pair of sex chromosome, and sex chromosome is X, Y, and male is the XY type, and female is the XX type.As shown in Figure 8, as follows according to every pair of feature of karyomit(e) size order:

1-3 number is submetacentric chromosome:

4-9 number is maximum telochromosome;

10-18 number is less telochromosome:

19-26 number is minimum telochromosome:

X chromosome is maximum acrocentric chromosome,

Y chromosome is minimum kinetochore karyomit(e).

Table 2 Minxian County black-fur sheep karyomit(e) parameter (♀)

Karyomit(e) number	Relative length (%)	Arm ratio index (%)	Centromere index (%)	The kinetochore type
Karyomit(e) number	Relative length (%)	Arm ratio index (%)	Centromere index (%)	The kinetochore type	1	10.8±0.76	1.03±0.03	0.43±0.01	M
2	9.15±0.07	1.32±0.06	0.44±0.03	M	1	10.8±0.76	1.03±0.03	0.43±0.01	M
2	9.15±0.07	1.32±0.06	0.44±0.03	M	3	5.51±0.69	1.03±0.06	0.42±0.04	M
4	4.85±0.11			T	3	5.51±0.69	1.03±0.06	0.42±0.04	M
4	4.85±0.11			T	5	4.83±0.16			T
6	4.39±0.21			T	5	4.83±0.16			T
6	4.39±0.21			T	7	4.12±0.10			T
8	4.04±0.04			T	7	4.12±0.10			T
8	4.04±0.04			T	9	3.87±0.18			T
10	3.81±0.09			T	9	3.87±0.18			T
10	3.81±0.09			T	11	3.55±0.16			T
12	3.21±0.20			T	11	3.55±0.16			T
12	3.21±0.20			T	13	3.21±0.13			T
14	3.10±0.12			T	13	3.21±0.13			T
14	3.10±0.12			T	15	3.11±0.18			T
16	2.94±0.17			T	15	3.11±0.18			T
16	2.94±0.17			T	17	2.82±0.19			T
18	2.82±0.10			T	17	2.82±0.19			T
18	2.82±0.10			T	19	2.81±0.10			T
20	2.82±0.15			T	19	2.81±0.10			T
20	2.82±0.15			T	21	2.52±0.14			T
22	2.51±0.28			T	21	2.52±0.14			T
22	2.51±0.28			T	23	2.48±0.39			T
24	2.44±0.45			T	23	2.48±0.39			T
24	2.44±0.45			T	25	2.38±0.41			T
26	2.21±0.34			T	25	2.38±0.41			T
26	2.21±0.34			T	X	6.39±0.12			ST

X

5.98±0.34

ST

Six, isozyme separates

Method: adopt the discontinuous gradient polyacrylamide gel electrophoresis.

1, collecting cell suspends again with PBS, numeration;

2, clean.Wash cell 3 times with PBS, the centrifugal supernatant of abandoning;

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

4, cell suspension is moved on in the 1.5ml centrifuge tube, under 4 ℃ with 1 * 10 ⁴Rpm is centrifugal, and 2min collects supernatant liquor, and packing postposition-70 ℃ storage is standby;

5, supernatant liquor adds equivalent 40% sucrose solution, and 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, and cuts concentrated glue, after twice of distilled water rinsing, immerses 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 Minxian County black-fur sheep ear rim tissue fibroblast cell is respectively LDH1 from " anode " to " negative electrode ", LDH2, and LDH3, LDH4, LDH5, the enzymic activity power is followed successively by LDH4＞LDH3＞LDH2＞LDH5＞LDH1.As shown in figure 10, the malate dehydrogenase zymogram of Minxian County black-fur sheep 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.Concrete data see Table 3, table 4.

The relative mobility of table 3 LDH isozyme

Kind	LDH1(％)	LDH2(％)	LDH3(％)	LDH4(％)	LDH5(％)
Kind	LDH1(％)	LDH2(％)	LDH3(％)	LDH4(％)	LDH5(％)	The Minxian County black-fur sheep	57.14	45.05	40.65	28.57	20.88

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

Kind	sMDH(％)	mMDH(％)
Kind	sMDH(％)	mMDH(％)	The Minxian County black-fur sheep	28.57	72.85

Above-mentioned isozyme electrophoresis result shows that the somatocyte heritability of being cultivated is stable, and crossed contamination does not take place between the cell purity height.

LDH and MDH expression of gene are subjected to the gene and the dual control of metabolite, and the fast molecular weight of district's band of electrophoretic velocity own is little.If cell is contaminated, then can be different because of the mobility of each kind, the result just may occur than present more district's band of more number.Utilize Minxian County black-fur sheep vitro culture inoblast to carry out lactic dehydrogenase isozyme (LDH) and malate dehydrogenase (malic acid dehydrogenase) (MDH) pedigree analysis can obtain feature pedigree clearly, show that the somatocyte heritability of being cultivated is stable, the purity height does not have the cell generation crossed contamination with other kinds.

Seven, the expression of pEYFP-N1 in culturing cell and the foundation of positive cell strain

Method:

1, the preparation of fluorescin reporter plasmid pEYFP-N1 and evaluation: extract the box specification sheets according to Plasmid Midi Kit plasmid, extraction, purifying 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 pEYFP-N1 fluorescin plasmid concentration that obtains is 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 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 513nm excitation, observing yellow fluorescence protein, calculate transfection efficiency.

3, apoptotic detection

(1), cell is inoculated in 12 well culture plates every hole 3ml, 37 ℃, 5%CO with 4.0 * 105/ml density ₂Be cultured to cell attachment in the 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.

4, the screening of transfection resistant cell

(1), Minxian County black-fur sheep ear rim tissue fibroblast cell is gone in the 25mL culturing bottle, every bottle adds the full nutrient solution of 5mL cell, treat that cell covers with, add G418:0,100,200,300,400,500,600,700,800g/mL by following concentration, continue to cultivate, observation of cell growth and death condition were changed liquid 1 time in per 3 days, still add G418, make all dead Cmin of cell be minimum lethal dose in two weeks with above-mentioned concentration.Determine that concentration is 800g/mL.

(2), behind the transfection 48h, cell is gone down to posterity by 1: 4 density, continue to be cultured to cell density and reach the 70%-80% fusion, abandon nutrient solution, the full nutrient solution of G418 of changing concentration 800g/mL screens, and changes nutrient solution one time in per 3 days, behind about 10d, big death of control cells, the formation number and the luciferase expression intensity of observing cell clone in every hole.G418 nutrient solution concentration is changed to 300g/mL and continues screening and culturing, obtains the stable clone of G418 resistant cell substantially.Select the strongest individual cells of luciferase expression clone and go to the amplification of further cultivating, go down to posterity of 50mL culturing bottle.

4, mono-clonal positive cell strain fluorescence protein gene integration, detection and the evaluation expressed

From the Minxian County black-fur sheep ear rim tissue fibroblast cell culture of the yellow fluorescence protein positive expression of selecting enlarged culturing, extract genomic dna, the positive RNA of yellow fluorescence protein adopts Trizol reagent to extract, the reverse transcription of mRNA is according to cDNA synthetic agent box, and the method for cDNA PCR library test kit specification sheets is carried out.Designed special primer P1 (5 '-TTCTCGCCACCATGGTGAGCAA-3 '), P2 (5 '-TTCTCGCGGCGGCCGCTTTACTTGT-3 ') 6 kinds of fluorescin positive cell genomic dnas of amplification, with with a pair of primer yellow fluorescence protein positive cell RNA being carried out RT-PCR, primer 18S51 (5 '-GGCAGCGTCCGGGAAACCAAATTC-3 ') and 18S31 (5-CCACCACAGATCAGAGAGC-3 ') amplification 18sRNA is as the RT-PCR internal reference.PCR reaction cumulative volume is 25 μ l, the PCR program: 95 ℃ of thermally denature 5min, and then with 94 ℃, 30s; 57 ℃, 30s; 72 ℃, after 30s carried out 35 circulations, the agarose gel electrophoresis with 1% detected the integration and the expression of fluorescence protein gene.

Conclusion:

Shown in Figure 11,12, the transfection peak of Minxian County black-fur sheep ear rim tissue fibroblast cell appears at 48h after the transfection, and transfection efficiency is 63.5%; Positive cell and part positive cell fluorescence intensity all reduce gradually, weaken behind the transfection 72h.The ratio of cell with 1: 4 gone down to posterity, after the 3～4h that goes down to posterity treats cell attachment, begin with G418 screening, screened for 2 weeks after, among a plurality of clones of acquisition, some still can see luciferase expression, some be can't see, and selects expression intensive clone and has carried out enlarged culturing, passes for 8～13 generations at present, these cells are still expressed fluorescence, and fluorescence intensity is still strong.Utilize design fluorescence protein gene special primer, the pEYFP-N1 fluorescin positive cell genomic dna that extracts is carried out pcr amplification, obtained the fragment about a 750bp, the clip size that increases with the primer expection of design just conforms, and illustrates that fluorescence protein gene all has been incorporated on the genome karyomit(e) of cell.With with a pair of primer, the cDNA that the pEYFP-N1 fluorescin positive cell RNA reverse transcription of extracting is obtained increases, also obtained the fragment about 750bp, confirmed on the rna level that fluorescence protein gene can transcribe normally, express in the black-fur sheep ear rim tissue fibroblast cell of Minxian County.Successfully obtained to be incorporated into the pEYFP-N1 fluorescence protein gene positive cell strain on the nuclei dyeing colour solid.As Figure 12.

Experimental results show that the pEYFP-N1 reporter gene in this ear margin tissue fibroblast line of Minxian county black fur sheep cell expressing rate far above 30～40% of other clone, expression rate is all more than 60%.This result of study has important value for researchs such as marker gene, nuclear transplantation and transgenic animal clones.

Claims

1, a kind of high motility rate, high purity ear margin tissue fibroblast line of Minxian county black fur sheep is characterized in that, its deposit number is CGMCC No.1880.

2, the cultural method of a kind of high motility rate, high purity ear margin tissue fibroblast line of Minxian county black fur sheep is characterized in that, this method comprises the steps:

(1) first culture: Minxian County black-fur sheep ear-edge tissue is cut into 1mm with after the PBS rinsing 6～8 times with eye scissors ³About tissue block; Tissue block is moved in the inversion culturing bottle, and at 37 ℃, 5%CO ₂CO ₂Cultivate 2～3h in the incubator; Treat that the complete adherent back of tissue block adds nutrient solution 8～10ml, the superfine foetal calf serum of nutrient solution composition: DMEM+10% continues to cultivate 10～12h;

(3) cell cryopreservation:

3, the cultural method of a kind of high motility rate according to claim 2, high purity ear margin tissue fibroblast line of Minxian county black fur sheep is characterized in that, the Minxian County black-fur sheep organization material described in the step (1) is the best with the children age.

4, the cultural method of a kind of high motility rate according to claim 2, high purity ear margin tissue fibroblast line of Minxian county black fur sheep, it is characterized in that, trysinization concrete steps described in step (2) and the step (3) are: add 0.25% trypsinase, 1～2ml in culturing bottle, be inverted the digestion 30～60s that overturns culturing bottle is preheated to 37 ℃ in incubator after.

5, the cultural method of a kind of high motility rate according to claim 2, high purity ear margin tissue fibroblast line of Minxian county black fur sheep, 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 1min continuously, cell moved in the culturing bottle be added with nutrient solution piping and druming and evenly place 37 ℃, 5%CO ₂Incubator in cultivate and get final product.