CN101012450B - Mount Wuzhi Pig ear rim tissue fibroblast series and culturing method thereof - Google Patents

Abstract

The invention discloses a high-living rate and high-purity mature fiber cell system of Mongolian horse (CGMCC No.1879) in the cellar biological domain, which is characterized by the following: culturing high-purity phoroblast without epithelial cell; maintaining the living rate of frozen cell between 93.2% and 98.7%; fitting for large scale of culturing.

Description

Mount Wuzhi Pig ear rim tissue fibroblast series and cultural method thereof

Technical field

The present invention relates to a kind of clone and cultural method thereof, specifically utilize the WZSP ear-edge tissue to carry out first culture, go down to posterity and cultivate the high motility rate of acquisition, high purity mount Wuzhi Pig ear rim tissue fibroblast series, belong to the cytobiology field.

Background technology

The livestock and poultry genetic resources is the important biomolecule resource that human society is depended on for existence and development, is the great strategic basic resource of country, is related to national security and sovereignty, has become one of world's " biological economy " epoch competition focal point.

In recent years, because the continuous deterioration of habitable environment, biological germ plasm resource faces serious crisis, directly threatens human existence and development.Statistic data according to Food and Argriculture OrganizationFAO (FAO) nineteen ninety-five shows: in the area, Central Africa of Sub-Sahara 738 livestock and poultry species are arranged approximately, and 15% be resource in imminent danger nearly, African geographic form is very severe.From nineteen ninety-five so far, be in endangered domestic animal and rise to 19% by 8%, poultry has risen to 34% by 20%.

The Asian-Pacific area has the whole world 1/5 livestock and poultry resource, and 1251 livestock and poultry species are registered, and is the distribution area of yak and buffalo in the world; It also is area with Russia duck, pheasant, quail, 1/3 pig variety and the distribution of 1/4 goat kind of nearly half.In the kind that is write down, have 10% to be resource in imminent danger approximately, and in the period of nineteen ninety-five to 1999, the endangered domestic animal in Asia rises to 14% by 11% according to statistics, poultry rises to 37% by 32%.

Ox, goat, sheep, pig, duck and the turkey kind in the whole world about 1/4 are distributed in Europe, and nearly 1/2 horse, chicken and goose are arranged.Under economic pressures, some kinds are eliminated by market because productivity is low, make it be in endangered danger, and for example the production of bird and pig only relies on a few kind.This situation is even more serious in the area, Eastern Europe, and unsettled politics cause has also quickened the extinction of many kinds.In by 2576 kinds that FAO write down, almost half is considered to resource in imminent danger.In several years of nineteen ninety-five to 1999 year, be in endangered domestic animal and rise to 49% by 33%, poultry rises to 76% by 65%.

In Latin America, existing kind have 20% considered to be in imminent danger.The bird that is in Critical Condition by 5% of nineteen ninety-five sharply rise to 1999 45%.Preservation to resource in imminent danger is extremely urgent.In geographic 571 kinds that are registered in the Near East, there are 44 to be considered to be in Critical Condition.In the North America, production intensification makes foodstuff production only rely on the minority kind.Therefore increased the weight of the multifarious crisis of livestock and poultry genetic resources.In 259 kinds that are recorded, have 35% endangered.

Hence one can see that, and world's livestock and poultry germ plasm resource is more and more narrower, and redemption endangered breed, preservation germ plasm resource have become the task of top priority.Because the diversified geography of China, ecology, weather condition, numerous national and different living habit for a long time through the raising and train and seed selection meticulously of numerous laborers, have formed the livestock and poultry genetic resources numerous, rich and varied, that characteristic is different in addition.According to the eighties in 20th century whole nation livestock and poultry species resource investigation, China has surplus the livestock and poultry species resource 570 (account for world's livestock and poultry species resource 15%), and 282 are indexed in " Chinese livestock and poultry species will ".China's livestock and poultry species resource has the characteristic of wide adaptability, unique properties, product excellence, enjoys great prestige both at home and abroad.For example Qin Chuan ox, Jin Nanniu, Nanyang ox, western Shandong ox etc. are the fine provenances of central plain area's development beef raising; The cashmere that Liaoning down producing goat, Inner Mongolia White Cashmere Goat etc. are produced, fine hair jewelry, expensive clothing and other valuables, the fast-selling market of its fabric; The cold sheep of Taihu Lake pig and little tail oestrused with the four seasons and breeding potential is high is celebrated; Originate in the sheep known for its fine thick wool and the Chungwei in desert semi-desert grassland district, Ningxia, the fur coat that produces attractive in appearance magnificent; Beijing duck is the raw material of baking fried pork stomach ﹠ chicken gizzard; Fine breed of chicken with thick brownish feathers delicious delicacy in Beijing once was a raw material of making the imperial court chicken; WZSP has that body is little, thin lean, the characteristics such as lean meat is many of genetic stability, skin, is the improved seeds that laboratory animal and life science aspect are selected for use; Utilize " WUJIBAIFENG WAN " of silkiefowl black bone processing, have good medicinal characteristic; China's cockfighting, pony have become companion's kind of people's amusement and recreation.

Because be subjected to the restriction of factors such as geography, traffic, the germplasm characteristic of some livestock and poultry genetic resourcess is not familiar with fully, its good characteristic remains further to be excavated.Simultaneously, along with the variation of people's consumers demand, still have a large amount of unpredictalbe good characteristics.In a word, China's livestock and poultry genetic resources is not only the important livestock industry production means, and is valuable human natural heritage, and they will play a significant role to social progress to human life quality's raising.Although there is the livestock and poultry species resource of numerous characteristic goods in China, most kinds that still belong to low production level.The output of live-stock product can not satisfy the demand of population growth and urban and rural residents' level of consumption raising far away.And in the evolution that the livestock industry production level improves; some local variety are cultivated kind or cross-fertilize seed gradually and are replaced; cause the local variety colony quantity with rich and varied hereditary property to reduce, some kinds are in imminent danger or endangered, and the part kind is become extinct.The end of the seventies and the beginning of the eighties, China's livestock and poultry species resource investigation result confirmed, existing 10 the local breedings of China disappear, 8 endangered, 20 quantity reduce.The enforcement period of the ninth five-year plan investigation shows, because factors such as a large amount of introductions of exotic species, the deterioration of the ecological environment, human undue utilization, the situation of livestock and poultry germ plasm resource is sorrow very, livestock and poultry species in imminent danger has reached 36, and 93% pig, 44% horse donkey, 35% ox, 20% poultry, 15% continuous goat germ plasm resource are subjected to threat in various degree.If importance to the livestock and poultry genetic resources, potential value understanding to local livestock and poultry resource is not enough especially, this trend is along with the raising of livestock industry production level will further aggravate, and its consequence causes the variation of livestock and poultry germ plasm resource more and more littler, has a strong impact on the Sustainable development of China's livestock industry.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.

WZSP is one of rare local pig breed, originate in mountain area, the Wuzhi Mountain, Hainan Province, have small, chest is narrower, back of the body waist is straight, belly is not sagging, buttocks is undeveloped, four limbs are thin and grow, the careful compactness of physique, little and grow, little upright, sharp-tongued, the mouth tube of ear little curved, by the big portion of hair be black, belly with four bursts of inboards be white, worship and mao be black or feature such as brown.The sharp length of, head little, flexible because of its body, build are commonly called as " mouse pig " like mouse.Its Fresh ﹠ Tender in Texture, thick flavor, Kui quinoline acid in the meat, propanedioic acid ethyl pentyl group fat content are higher than 8 to 20 times of market pork respectively, aminoacids content also contains three-type-person's body indispensable amino acid etc. up to 72.615% in the meat, is important component part in the Chinese Pigs kind genetic diversity.Only deposit 100,000 the sixties in last century, nineteen eighty-two livestock on hand 600 remainders, investigation in 1987 finds only to have 10 remainders purebred, and is endangered.At present, the development of cryogenic freezing preservation technology and perfect, make any vitro culture thing to be preserved indefinitely on the basis of its vigor keeping, for saving this kind, the best approach is exactly to fibroblastic large scale culturing of WZSP and in addition preservation.

At present, normal employing Epstein-Barr virus conversion method, adherent culture method and enzyme digestion carry out the livestock and poultry cells in vitro and cultivate in a large number, but comparative test result shows: the Epstein-Barr virus conversion method is cultivated for the cell in vitro of domestic animals kind and is inapplicable, and its cultured cells motility rate and purity all can not reach required standard.The primary cell that enzyme digestion 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 domestic animals cell, cell purity and activity all have higher requirement, and at present, also there are some shortcomings in the adherent culture method, and be single as method, cost is too high, cytoactive and purity is on the low side, cell mortality ratio behind cryopreservation resuscitation is high excessively.Therefore, be badly in need of its method is improved so that the WZSP inoblast reaches the highly purified standard of high cell motility rate, and can obtain good preservation and continue.

Summary of the invention

The purpose of this invention is to provide mount Wuzhi Pig ear rim tissue fibroblast series, its preserving number is: CGMCCNo.1879.

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 reactivity, highly purified mount Wuzhi Pig ear rim tissue fibroblast series, and in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, 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: CGMCCNo.1879, the classification called after mount Wuzhi Pig ear rim tissue fibroblast series of suggestion.This WZSP ear-edge tissue embryo fibroblast has following feature: fast growth, motility rate height, exogenous gene expression rate height, applied range.

The cultural method of mount Wuzhi Pig ear rim tissue fibroblast series, this method comprises the steps:

(1) first culture: the WZSP 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 of nutrient solution composition: DMEM+10%.

(2) cultivation of going down to posterity: the nutrient solution in reject step (1) culturing bottle, carefully wash in the culturing bottle residue 2 times with the PBS that is preheated to 37 ℃, to remove remaining serum, the tissue block that comes off and dead cell.Stop digestion with adding nutrient solution 6～10ml behind the tryptic digestion; Postdigestive cell 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, remove supernatant liquor, the frozen storing liquid 1ml that adds 4 ℃ of precoolings, blowing and beating gently with suction pipe behind the mixing makes cell resuspended, blow and beat gently with suction pipe and to make cell resuspended, and shorten shelf-time in the chamber of frozen storing liquid and cell (the frozen storing liquid composition: the 10%DMSO+50% foetal calf serum+40%DMEM); as far as possible

E, packing: the cell packing is gone in the frozen pipe of sterilization to seal, indicate date, kind, cell title, cultivate algebraically;

F, pre-freeze: with frozen pipe load program the cooling box in, be put in 4 ℃ of 20～30min, DMSO fully is penetrated in the cell, reach internal and external equilibrium, place then more than-70 ℃ of refrigerator pre-freeze 4h.

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

Above-mentioned is the cultural method of high motility rate, high purity mount Wuzhi Pig ear rim tissue fibroblast series, wherein, cell frozen in the step (3) can be carried out cell recovery according to actual needs, concrete steps are: insert rapidly in 42 ℃ of water-baths after frozen pipe is taken out from liquid nitrogen, rock about 1min fast, cell moved in the culturing bottle be added with nutrient solution piping and druming evenly, place and contain 37 ℃ of 5%CO ₂Incubator in continue to cultivate.

Advantage of the present invention and benefit: the present invention adjusts and improves 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 compared with prior art has significantly raising; After improving frozen condition, 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, and the cell motility rate behind the cell cryopreservation can reach 93.2%～98.7%, and the passage growth is stable, compare with existing cell culture technology and to be significantly increased, be fit to large scale culturing.The adjustment of substratum and the improvement of cultural method have significantly reduced cost.The present invention has remedied the deficiency that existing domestic animal somatocyte is preserved in each side such as methods, and the genetic resources that the cell motility rate of mount Wuzhi Pig ear rim tissue fibroblast series and the lifting of purity also make WZSP kind important, in imminent danger is able to prolonged preservation with the form of cultured cell in vitro.

Because the scarcity of WZSP germ plasm resource makes that people's research to WZSP in each field is few.High motility rate of the present invention, high purity mount Wuzhi Pig ear rim tissue fibroblast series can provide a large amount of high quality material for life sciences such as genetically engineered, cell engineering, immunology and molecular biology; Also can be used as the donorcells of domestic animal somatic cell clone breeding; On agricultural, can enrich and improve local variety, can be used as one of guarantor's kind means of local improved seeds simultaneously; Also can be used as the main raw material(s) of production of vaccine.

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 WZSP ear-edge tissue primary cell microscope figure below;

Fig. 2 is preceding microcytoscope figure below for the WZSP ear-edge tissue goes down to posterity;

Fig. 3 is WZSP ear-edge tissue subculture I microcytoscope figure below;

Fig. 4 is WZSP ear-edge tissue subculture II microcytoscope figure below;

Fig. 5 is by the WZSP ear rim tissue fibroblast cell of mycoplasma contamination contrast figure;

Fig. 6 is a WZSP ear rim tissue fibroblast cell detection of mycoplasma negative findings diagram;

Fig. 7 is WZSP ear rim tissue fibroblast cell growth curve figure;

Fig. 8 is a WZSP ear rim tissue fibroblast cell caryogram diagram;

Fig. 9 is a WZSP ear rim tissue fibroblast cell lactic dehydrogenase isozyme electrophoresis diagram;

Figure 10 is a WZSP ear rim tissue fibroblast cell malate dehydrogenase (malic acid dehydrogenase) electrophoresis diagram;

Figure 11 is that foreign gene is expressed 48h diagram (transfection of pEGFP-N3 fluorescin plasmid) in WZSP ear rim tissue fibroblast cell;

Figure 12 is that foreign gene is expressed 72h diagram (transfection of pEGFP-N3 fluorescin plasmid) in WZSP ear rim tissue fibroblast cell;

Figure 13 is that foreign gene is expressed 72h diagram (transfection of pDsRed1-N1 fluorescin plasmid) in WZSP ear rim tissue fibroblast cell;

Figure 14 is that foreign gene is expressed 72h diagram (transfection of pEYFP-N1 fluorescin plasmid) in WZSP ear rim tissue fibroblast cell

Figure 15 is two cell stages of the xenogenesis nuclear transplantation of donor for WZSP ear rim tissue fibroblast cell

Figure 16 is the morula embryos of the xenogenesis nuclear transplantation of donor for WZSP ear rim tissue fibroblast cell

Figure 17 is the blastula embryo of the xenogenesis nuclear transplantation of donor for WZSP ear rim tissue fibroblast cell

Embodiment

Used key instrument and reagent source among the embodiment:

WZSP ear-edge tissue source: mountain area, the Wuzhi Mountain, Hainan Province.

(1) key instrument equipment

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

2, cytogamy instrument: U.S. BTX ECM-2001

3, micrurgy instrument: Japanese Nikon NT-88-V3

4, embossment phase microscope, Olympus IX-71; Biomicroscope, Olympus CX31; 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, CO ₂Incubator: 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, carrier pEGEP-N3: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 (DsRed and 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

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 (pH 8.0) 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 (pH 8.0)

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: 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.

43, cytochalasin B liquid (CCB): the cytochalasin B of 1mg is dissolved among the DMSO of 0.5mL, and concentrated solution is made in filtration sterilization, the time spent in 10ml NCSU-23 liquid, add this liquid 37.5 μ L and CCB liquid.

44, the Hoechst33342 of 5 μ g/mL Hoechst33342:0.005g is dissolved in the 10ml water, and filtration sterilization is made and added the above-mentioned concentrated solution of 5 μ L in the concentrated solution times spent 500 μ L nutrient solution.

45,5 μ M/L ionomycins activate: the ionomycin of 1mg is dissolved among the DMSO of 0.5mL, and concentrated solution is made in filtration sterilization, and the time spent is added among the NCSU-23.

46, the 6-DMAP of 2mM/L: the 6-DMAP that takes by weighing 0.0065g is dissolved among the NCSU-23 of 20mL.

47, NCSU-23 embryo medium: NaCL 0.6355g, NaHCO ₃0.2106g, KCL 0.0355g, glucose 0.1g, potassium primary phosphate 0.0162g, sal epsom 0.0293g, Calcium dichloride dihydrate 0.025g, taurine 0.0879g, hypotaurine 0.0546g.

The structure of embodiment 1 high motility rate, high purity mount Wuzhi Pig ear rim tissue fibroblast series

(1) first culture: the WZSP 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 of nutrient solution composition: DMEM+10%.

(2) cultivation of going down to posterity: the nutrient solution in reject step (1) culturing bottle, carefully wash in the culturing bottle residue 2 times with the PBS that is preheated to 37 ℃, to remove remaining serum, the tissue block that comes off and dead cell.Stop digestion with adding nutrient solution 6～10ml behind the tryptic digestion; Postdigestive cell 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, remove supernatant liquor, the frozen storing liquid 1ml that adds 4 ℃ of precoolings, blowing and beating gently with suction pipe behind the mixing makes cell resuspended, blow and beat gently with suction pipe and to make cell resuspended, and shorten shelf-time in the chamber of frozen storing liquid and cell (the frozen storing liquid composition: the 10%DMSO+50% foetal calf serum+40%DMEM); as far as possible

E, packing: the cell packing is gone in the frozen pipe of sterilization to seal, indicate date, kind, cell title, cultivate algebraically;

F, pre-freeze: with frozen pipe load program the cooling box in, be put in 4 ℃ of 20～30min, DMSO fully is penetrated in the cell, reach internal and external equilibrium, place then more than-70 ℃ of refrigerator pre-freeze 4h.

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

(4) cell recovery: insert rapidly in 42 ℃ of water-baths after frozen pipe taken out from liquid nitrogen, rock fast about 1min, cell is moved in the culturing bottle that is added with nutrient solution piping and druming evenly, place and contain 37 ℃ of 5%CO ₂Incubator in continue to cultivate.

The detection of embodiment 2 mount Wuzhi Pig ear rim tissue fibroblast series biological characteristicses

The mount Wuzhi Pig ear rim tissue 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 (WZSP primary cell), Fig. 2 (WZSP go down to posterity preceding cell), Fig. 3 (WZSP subculture 1 cell), Fig. 4 (WZSP 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 WZSP fibroblastic soybean Tryptones nutrient solution and wort nutrient solution, 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: conventional cultivation WZSP inoblast 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 film-making of vitro culture WZSP inoblast, 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 WZSP 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 mount Wuzhi Pig ear rim tissue fibroblast series 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 PDT35.9h 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 WZSP 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 WZSP 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 mount Wuzhi Pig ear rim tissue fibroblast series 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).

WZSP diploid chromosome number order is 2n=38, and totally 19 pairs, sex chromosome is X, Y, and male is the different XY that joins, and is female for joining XX together.As shown in Figure 8, karyological character is described below respectively:

1～No. 5: submetacentric chromosome;

6～No. 7: acrocentric chromosome, size are only second to karyomit(e) No. 1;

8～No. 12: metacentric chromosome;

13～No. 18: telochromosome;

Table 2 WZSP karyomit(e) parameter (♂)

Annotate: karyomit(e) is pressed the arm ratio index and divided: 1.0～1.6 are metacentric chromosome (M), and 1.7～2.9 is submetacentric chromosome (sM), and 3.0～6.0 is acrocentric chromosome (ST), and index＞7.0 are telochromosome (T).

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:

The LDH of table 3 WZSP cell and MDH isozyme relative mobility

As shown in Figure 9, WZSP ear-edge tissue acid dehydrogenation isozyme zymogram is respectively LDH1 from " anode " to " negative electrode ", LDH2, and LDH3, LDH4, LDH5, the enzymic activity power is followed successively by LDH5, LDH4, LDH3, LDH2, LDH1.As shown in figure 10, the malate dehydrogenase zymogram of WZSP 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.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.LDH and MDH analyze can obtain feature pedigree clearly, shows the somatocyte purity height of being cultivated, and heritability is stable, with the iuntercellular of other kinds crossed contamination does not take place.

Seven, the expression of foreign gene in 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, use the trypsin digestion and cell culture, with the cell inoculation under the digestion in the culture plate of 6 holes (or 35mm), every hole 1～2 * 10 ⁵Cell is added 2ml and is contained the serum nutrient solution;

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

(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 is with twice in the nutrient solution rinsing cell that does not contain 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 culture plate, 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 fibroblastic transfection of WZSP peak appeared at transfection 48h, for the fluorescence intensity of green fluorescence cell reaches the byest force, transfection efficiency was 64.5%; Yellow fluorescence protein pEYFP-N1 transfection efficiency behind transfection 48h has reached 56.2%, as shown in figure 14; Red fluorescent protein pDsRed1-N1 transfection efficiency behind transfection 48h has reached 68.8%, as shown in figure 13; Positive cell and fluorescence intensity all reduce gradually, weaken behind the transfection 48h.Show that this clone exogenous gene expression rate is far above other clone exogenous gene expression rate 30～40%.

Eight, the detection of the donorcells of growing as xenogenesis nuclear transplantation reconstituted embryo

(1), select the ripe bovine oocyte of discharging first polar body, cytochalasin B (CCB) is hatched 15min;

(2), sucking-off polar body and near parts of fine kytoplasm carry out stoning together with Metaphase Chromosome;

(3), 5 μ g/mL Hoechst33342 balance 10min, fluorescent microscope is selected complete non-nucleus egg mother cell;

(4), the WZSP inoblast behind the 0.25% tryptic digestion serum starvation 3d is as donorcells, donorcells injected that the bovine oocyte in complete stoning constructs reconstituted embryo by electricity under the zona pellucida by micrurgy;

(5), after the reconstituted embryo that builds activates 5min with 5 μ M/L ionomycins, move in the 6-DMAP nutrient solution that contains 2mM/L and activate 4h again, the reconstituted embryo after the activation is put into four well culture plates (Nunc) and is cultivated, and cultivates the blastaea number of statistics growth behind 7～8d;

(6), the karyotyping step of reconstituted embryo is the same.

Conclusion: wherein donorcells is one of critical elements of body-cell neucleus transplanting, and its state is to influence the important factors that reconstituted embryo is grown.Donorcells is carrying all genetic material that late embryogenesis is grown, and finishes this process of reprogramming in enucleation oocyte.

As donor nuclei, the ox enucleation oocyte makes up xenogenesis nuclear transplantation reconstituted embryo as recipient cytoplasm with the WZSP inoblast, check WZSP inoblast developmental potency.The result as shown in the figure, Figure 15 for WZSP ear rim tissue fibroblast cell be two cell stages of the xenogenesis nuclear transplantation of donor; Figure 16 is the morula embryos of the xenogenesis nuclear transplantation of donor for WZSP ear rim tissue fibroblast cell; Figure 17 is the blastula embryo of the xenogenesis nuclear transplantation of donor for WZSP ear rim tissue fibroblast cell.This shows, the WZSP inoblast can obtain sequencing again as donorcells in the ox enucleation oocyte, the nuclear transplantation reconstituted embryo blastaea rate (34%) that the WZSP fibroblast that present method obtains makes up is compared than the motility rate 7.3%～20% of the nuclear transplantation reconstituted embryo of prior art culturing cell structure and is increased significantly,, consistent through the genetic material of karyotyping decidable reconstituted embryo with the karyomit(e) type of nuclear donor cell from the WZSP somatocyte.

Claims (1)

1. mount Wuzhi Pig ear rim tissue fibroblast series is characterized in that, deposit number is CGMCCNo.1879.

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