CN102382799A - In-vitro isolation and culture method of mouse spermatogonial stem cells and special culture medium thereof - Google Patents
In-vitro isolation and culture method of mouse spermatogonial stem cells and special culture medium thereof Download PDFInfo
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Abstract
The invention provides an in-vitro isolation and culture method of mouse spermatogonial stem cells, comprising the steps of: step 1, collecting a mouse testicle, and adopting a two-step enzymic digestion method to prepare a cell suspension; step 2, adopting an immunomagnetic bead sorting method to isolate the spermatogonial stem cells; step 3, preparing a culture solution of the spermatogonial stem cells, and implementing primary culture and subculture for the spermatogonial stem cells. The invention also provides a culture medium adopted by the method; the culture medium consists of an STO cell feeding layer and the culture solution of the spermatogonial stem cells; the culture solution of the spermatogonial stem cells comprises a basic medium, an FBS (Fetal Bovine Serum), an amino acid, an LIF (Leukemia Inhibitory Factor), a bFGF (Basic Fibroblast Growth Factor), an IGF-IR (Insulin-like Growth Factor-I Receptor) activator, a vitamin C, an EGF (Epidermal Growth Factor )and a GDNF (Glial cell line-Derived Neurotrophic Factor). By adopting the method and the culture medium disclosed by the invention, the spermatogonial stem cells sourced from a larval mouse or an adult mouse can be cultured and proliferated in vitro for a long term, a spermatogonial stem cells line which can keep a stable growth for the long term can be constructed, and a sperm generation function of the spermatogonial stem cells can be maintained. The in-vitro isolation and culture method of the mouse spermatogonial stem cells disclosed by the invention has the advantages of convenient operation, high safety and reliability and low cost.
Description
Technical field
The present invention relates to the in-vitro separation cultural method and the special culture media thereof of spermatogonial stem cells into mouse.
Background technology
Stem spermatogonium is the undifferentiated sperma-togonium A of part, is positioned at boar convoluted tubule of testis seminiferous epithelium basement membrane, occupies extremely important status in complicacy in the spermatogeny process of high-sequential.Stem spermatogonium is a kind of stem cell that can gene be passed to the offspring, and normal self of stem spermatogonium and propagation, differentiation have guaranteed lifelong sexual cell supply in male creature.Stem spermatogonium is kept certain number and kept normal propagation, differentiation function is the key of guaranteeing male fertility.Under suitable environment and condition, stem spermatogonium can be divided into other cells of tissues.
The research of stem spermatogonium and application need obtain long-term cultivation and keep the stable cell lines of differentiation capability in its body; The cultivation of these stable cell lines mostly comes from the testis tissue of mouse childhood, Sun Yuan etc. " foundation [J] of spermatogonial stem cells into mouse vitro culture system. Chinese andrology magazine, 2008; 14 (8): 695～700 " disclosed following content in: 30 of the male ICR mouses of the living back of taking-up 2～6d; Take off the neck method and put to death, cut testis after, adopt two step enzyme digestions to process single cell suspension; Add specific nutrient solution, carry out vitro culture as feeder layer cells with MEC.Culturing cell is identified; The result: spermatogonial stem cells into mouse is in external stable propagation; The DCRP AP of institute strong positive; Markers tests GFR α-1+/Oct-4+/VASA+/SCP3-, genetic expression GFR α-1+/Oct-4+/SCP3-confirms that institute's culturing cell is the spermatogonial stem cells into mouse that is in undifferentiated state.Spermatogonial stem cells into mouse can be stablized and goes down to posterity and keep undifferentiated state under the culture system that this research is set up, and for exploring the external sperm generating process good research platform is provided." nature " magazine was rolled up the 1199th～1203 page of the 7088th phase at 2006 the 440th and has been published " Pluripotency of spermatogonial stem cells from adult mouse testis " (multipotency of adult mice stem spermatogonium) literary composition; Mention in the literary composition that the individual stem spermatogonium of growing up can develop into various tissues.
Literature search through to prior art is found; " mutation research " (Mutat.Res.) magazine published the articles of a piece " A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse " by name (quantitative examination of stem spermatogonium self in the C3H/101 F1 hybridization mouse) 193～200 pages of 1993 the 290th phases; Mention in the literary composition; In juvenile; Approximately in per 10,000 testicular cells 2～3 stem spermatogoniums are arranged, only account for 0.02%～0.03% of all seminiferous epithelium TCSs of testis, and the shared ratio of stem spermatogonium is lower in the adult testis; Differentiation degree is higher, thereby the difficulty of long-term cultivation is bigger.Set up clone and produce the offspring mouse with the adult mice stem spermatogonium, all do not see relevant report at home and abroad.Therefore, the present invention's technical problem that will solve is: how at vitro culture adult mice stem spermatogonium.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of in-vitro separation cultural method of spermatogonial stem cells into mouse is provided.Utilize method of the present invention, can make the stem spermatogonium that derives from the young or adult mouse all can set up the stem spermatogonium system of long term maintenance stable growth, and keep its spermatogenetic function in external long-term cultivation and propagation.
A kind of in-vitro separation cultural method of spermatogonial stem cells into mouse comprises the steps:
In the step 1, the magnetic bead that said immunological magnetic bead sorting method is used is the Thy1 magnetic bead.
In the step 1, said Thy1 magnetic bead is CD90.
In the step 3, adding STO cell feeder layer when cultivating is supported and gone down to posterity to said former being commissioned to train, and the preparation method of said STO cell feeder layer is specially: in the STO cell, add the STO cell culture fluid that contains 10 μ l/ml ametycins, cultivate in incubator afterwards; The washing of PBS damping fluid; Add 0.25% trypsinase, place incubator to digest 4-5min; Add the STO cell culture fluid and stop tryptic digestion, and, cell is uniformly dispersed with liquid-transfering gun piping and druming; The transitional cell suspension is to centrifuge tube, and is centrifugal, abandoning supernatant; In centrifuge tube, add the STO cell culture fluid, blow even and fine born of the same parents, divide to the plate that is covered with gelatin then with liquid-transfering gun; Add the STO cell culture fluid; With the cell that liquid-transfering gun is handled the nutrient solution homodisperse, behind the cultivation 24h, the STO cell that obtains promptly can be used as the feeder layer of stem spermatogonium.
Said STO cell cultures liquid formula: 88%DMEM, 10%FBS, 0.3mg/ml L-glutaminate, 100U/ml penicillium mould.
In the step 3, the said cultivation of going down to posterity is specially went down to posterity 1 time in 4-7 days.
Another object of the present invention is also to provide a kind of employed substratum of in-vitro separation cultural method of spermatogonial stem cells into mouse; Be made up of STO cell feeder layer and stem spermatogonium nutrient solution, said stem spermatogonium nutrient solution comprises MEM-α, FBS, L-glutaminate, LIF, bFGF, Regular Insulin, vitamins C, EGF, GDNF.
Preferably, said stem spermatogonium nutrient solution further comprises more than one in non-essential amino acid, beta-mercaptoethanol, the Sodium.alpha.-ketopropionate.
Preferably, the volumn concentration of said FBS is 8-10%, and the content of said LIF is 10
2-10
4U/ml; The content of said bFGF is 0.02-0.03 μ g/ml, and the content of said IGF-1R activator is 0.04-0.06 μ g/ml, and the content of said EGF is 0.01-0.02 μ g/ml; Said ascorbic content is 40-60 μ g/ml, and the content of said GDNF is 0.01-0.02 μ g/ml.
Preferably, in said stem spermatogonium nutrient solution, the volume percent of said FBS is 10%, and the content of said LIF is 10
3U/ml, the content of said bFGF are 0.025 μ g/ml, and the content of said IGF-1R activator is 0.05 μ g/ml, and said ascorbic content is 50 μ g/ml, and the content of said EGF is 0.01 μ g/ml, and the content of said GDNF is 0.01 μ g/ml.
In the step 3; The concrete steps of preparing the nutrient solution of said stem spermatogonium are: being formulated as of 100ml stem spermatogonium nutrient solution: get 86.8ml MEM-α, 10ml FBS, 1ml 30mg/ml L-glutaminate; The 1ml non-essential amino acid; 1ml 100mmol/L Sodium.alpha.-ketopropionate, 0.7 μ l beta-mercaptoethanol, 100 μ l 10
6U/ml LIF, 5 μ l, 1 μ g/ μ l Regular Insulin, 5 μ l 1mg/ml vitamins Cs, 2.5 μ l, 1 μ g/ μ l bFGF, 1 μ l, 1 μ g/ μ l EGF, 1 μ l1 μ g/ μ l GDNF; Above each component mixing is promptly got.
Said non-essential amino acid is bought from Invitrogen company.
Compared with prior art; The present invention has following beneficial effect: utilize method of the present invention; Can make that the stem spermatogonium that derives from the young or adult mouse all can be in external long-term cultivation and propagation; Can set up the stable stem spermatogonium system of long term maintenance, and keep its spermatogenetic function; Simultaneously, method of the present invention is easy and simple to handle, and is safe and reliable, with low cost.
Description of drawings
Fig. 1: the photo of the foundation of stable spermatogonial stem cells into mouse system.(a) show the adult stem spermatogonium form (arrow shown in) of STO cell feeder layer after last 24 hour of cultivating in that ametycin was handled.(b) cultivate after 4 months typical stem spermatogonium bunch and clone's form.Scale: 10 μ m in a; 50 μ m inb.
Fig. 2: stem spermatogonium is transplanted the photo of the testis section after 12 weeks, sees spermatogeny.Scale: 40 μ m.
The adult mice stem spermatogonium system in Fig. 3: C57BL/6 source be implanted into C57BL/6 * CD-1 F1 for the acceptor mouse after, with the mating of purebred CD-1 mouse, and the offspring who produces.Mother is a pure white, and the offspring is black.
The immunofluorescence that Fig. 4: PCR detects STPB-C transgenic positive mouse detects photo; The M:DNA molecular weight standard; P: positive control; 2 and 7: the transgenic positive mouse; 1,3,4,5,6,8,9 and 10: wild-type mice.
The electrophoretogram of Fig. 5: Southern hybridization checking STPB-C transgenic positive mouse; The M:DNA molecular weight standard; P: positive control; N: negative control; 1 and 3: the transgenic positive mouse; 2,4,5 and 6: wild-type mice.
The immunofluorescence that Fig. 6: PCR detects STPB-C gene silencing mouse detects photo; The M:DNA molecular weight standard; P: positive control; 6,7,8,9 with 13:STPB-C gene silencing mouse; 1,2,3,4,5,10,11,12,14,15 and 16: wild-type mice.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Be interpreted as: these embodiment only be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; Sambrook equimolecular clone for example: laboratory manual is seen the condition described in the New York Cold Spring Harbor Laboratory press version in 1989, or the condition of advising according to manufacturer.
The foundation and the transplanting of spermatogonial stem cells into mouse system
With required childhood, adult or Aged Mice execution, clean the mouse health with 75% cotton ball soaked in alcohol; Cut open the belly rapidly and get bilateral testes, testis is placed the ice bath plate that contains 1/3 volume D-Hanks liquid approximately, and remove the coating of testis tissue; Other gets a sterilization 1.5ml EP pipe, adds 1ml D-Hanks liquid, and isolating seminiferous tubule is transferred to wherein; Get D-Hanks liquid that 3.5ml contains the 1mg/ml collagenase to the 15ml centrifuge tube, the D-Hanks liquid that will contain seminiferous tubule sucks wherein, and contains the D-Hanks cleaning and removing residual tissue of collagenase IV with 500 μ l, sucks together in the 15ml centrifuge tube, and making TV is 5ml; The 15ml centrifuge tube is placed 37 ℃ of water-baths, and slightly vibration digests 15min, after digestion finishes, and the centrifugal 5min of 1000rpm, abandoning supernatant; Add 5ml D-Hanks liquid in centrifuge tube, the centrifugal 5min of 1000rpm, abandoning supernatant is with method washing 3 times; Adding 5ml contains the D-Hanks liquid of 0.25% trypsinase and 0.02%EDTA, and 37 ℃ of digestion 10min that slightly vibrate then add 500 μ l FBS and stop digesting; The centrifugal 5min of 1000rpm, abandoning supernatant; In cell, add the Thy1 magnetic bead (CD90) (purchase) of 400ml immunological magnetic bead sorting BD buffer (damping fluid) (purchase) and 40 μ l, behind the mixing, place 30min gently in 6-8 ℃ of refrigerator from BD Biosciences company from BD Biosciences company; The 1.5ml centrifuge tube is fixed on the magnetic bead sorting shelf, adds mixed solution in centrifuge tube, on the magnetic bead sorting frame, place 6-8min then; Slowly siphon away BD buffer (damping fluid) mixed solution, take off this centrifuge tube (details are with reference to the product manual (protocol) of BD Biosciences company); The cell that adds the magnetic bead sorting in the resuspended centrifuge tube of 1.5ml stem cell nutrient solution changes cell over to Φ 35mm petridish or the cultivation of 24 orifice plates; After cultivating 3-4h, can examine under a microscope the somatocyte that mixes wherein begin adherent, the petridish that carefully tilts, with not adherent as yet spermatogonium together with nutrient solution (being the stem spermatogonium nutrient solution) sucking-off, inoculation culture separately.
1, the nutrient solution prescription of stem spermatogonium
The preparation of 100ml stem spermatogonium nutrient solution is specially: get 86.9ml MEM-α, 10ml FBS, 1ml30mg/ml L-glutaminate; 1ml non-essential amino acid (purchase) from Invitrogen company; The 1ml100mmol/L Sodium.alpha.-ketopropionate, 0.7 μ l beta-mercaptoethanol, 100 μ l 10
6U/ml LIF, 5 μ l, 1 μ g/ μ l Regular Insulin, 5 μ l 1mg/ml vitamins Cs, 2.5 μ l, 1 μ g/ μ l bFGF, 1 μ l, 1 μ g/ μ l EGF, 1 μ l, 1 μ g/ μ l GDNF; Above each component mixing is promptly got.
2, the preparation of STO feeder layer cells
(1) get some new Φ 60mm plates, add the 1ml gelatin respectively, room temperature is removed gelatin after Bechtop is placed 2h, continues culture plate is placed Bechtop, and is for use after drying;
(2) nutrient solution old in the STO culture dish is removed in suction; In the STO culture dish, add the STO nutrient solution 2ml (this nutrient solution need place incubator to carry out balance in advance) that contains 10 μ l/ml ametycins; Place incubator to handle 1.5h the STO culture dish, said STO cell cultures liquid formula is 88%DMEM, 10%FBS, 0.3mg/ml L-glutaminate and 100U/ml penicillium mould;
(3) behind the processing 1.5h, take out plate, remove nutrient solution, in the STO culture dish, add 1ml PBS damping fluid, rock the STO culture dish gently, discard the PBS damping fluid, so repeatable operation is 5 times;
(4) in the STO culture dish, add 1ml 0.25% trypsinase, place incubator to digest 4-5min; Add isopyknic fresh STO nutrient solution (not containing ametycin) and stop tryptic digestion, and, cell is uniformly dispersed with liquid-transfering gun piping and druming;
(5) the transitional cell suspension is to the 12ml centrifuge tube; The centrifugal 5min of 1000rpm, supernatant discarded adds 3ml STO nutrient solution (not containing ametycin) in centrifuge tube; Blow even and fine born of the same parents with liquid-transfering gun; Divide then to 2-4 is individual to be covered with in the described Φ 60mm of step (1) plate of gelatin, add fresh STO nutrient solution (not containing ametycin) to 4ml, and with the cell of liquid-transfering gun with the processing of nutrient solution homodisperse;
(6) the STO cell after the processing behind the cultivation 24h, promptly can be used as the feeder layer of stem spermatogonium.
3, the cultivation of stem spermatogonium, build and be
37 ℃, 5%CO
2Under the condition, on the STO feeder layer, cultivate stem spermatogonium, wherein the add-on of stem spermatogonium nutrient solution should determine according to the size of culture hole or petridish; Simultaneously, in order to keep the characteristic of this stem spermatogonium, in the nutrient solution of stem spermatogonium, also need add other special ESCs; Like LIF, Regular Insulin, EGF; GDNF, bFGF etc., concrete preparation sees the first step for details.Usually, in 24 well culture plates, every hole adds 500 μ l stem spermatogonium nutrient solutions, and in the Φ 60mm culture dish, needs to add 2ml stem spermatogonium nutrient solution, changes liquid 1 time in per 48 hours simultaneously, goes down to posterity 1 time in 5-7 days.
The passage method
Must prepare new STO feeder layer cells before going down to posterity, see the operation of the 2nd step for details.Carefully remove old stem spermatogonium nutrient solution, add 0.5ml 0.25% trypsinase and clean cell surface, remove trypsinase then; Add 1ml 0.25% trypsinase, place 37 ℃ of incubators, the time should not surpass 5min, and cell adds the fresh stem spermatogonium nutrient solution of 3ml and stops digesting when half comes off approximately by the time; Dispel cell gently with liquid-transfering gun, be transferred to centrifuge tube, the centrifugal 5min of 1000rpm, abandoning supernatant; Add the fresh stem spermatogonium nutrient solution of 2ml, dispel cell, divide then to the Φ 60mm plate of 2-4 STO feeder layer cells that contains new processing with liquid-transfering gun; Each plate adds fresh stem spermatogonium nutrient solution to 4ml, and with liquid-transfering gun nutrient solution is dispersed in whole plate.(a) of Fig. 1 shown the adult stem spermatogonium form (arrow shown in) of STO cell feeder layer after last 24 hour of cultivating in that ametycin was handled.(b) show to cultivate after 4 months typical stem spermatogonium bunch and clone's form.Scale: 10 μ m in (a); 50 μ min (b).
4, the transplanting of spermatogonial stem cells into mouse system
(1) preparation of acceptor mouse
To 4-6 male mice in age in week, press 40mg/kg dosage abdominal injection busulfan (Busulfan, Sigma company).After the injection, mixed fodder fed at least 4 weeks, and the survival mice testicular spermatogenic function is destroyed, and can be used as the acceptor mouse and carried out the spermatogonium transplanting.
(2) preparation of stem spermatogonium suspension
With 0.25% tryptic digestion culture (for having cultivated the cell in 40 generations), shift floating cell to centrifuge tube, 1, the centrifugal collection stem spermatogonium of 000rpm cell adds cold sterilization PBS damping fluid re-suspended cell, and the adjustment cell density is about 10
3/ μ l.
(3) transplanting of stem spermatogonium
Press 75mg/kg dosage, the abdominal injection Veronal sodium is anaesthetized male mouse.Clean the mouse health with 75% cotton ball soaked in alcohol, cut the abdominal cavity, extrude testis, the cell suspension multiple spot is injected in the mouse convoluted seminiferous tubule, note avoiding injuring blood vessel with glass capillary along near the ventrimeson.After injection finishes, testis is also gone into the abdominal cavity, sew up the incision.Transplant after two months, male mouse of postoperative and female mouse are mated.
The result shows and will transplant in infertile male mice convoluted seminiferous tubule from the stem spermatogonium of stem spermatogonium system that differentiation produces sperm in body, produces the mouse offspring with the female mice mating, sees Fig. 2,3.Fig. 2 has shown the testis section after stem spermatogonium transplanted for 12 weeks, and seeing has spermatogeny, scale: 40 μ m.Fig. 3 shown the adult mice stem spermatogonium system in C57BL/6 source be implanted into C57BL/6 * CD-1 F1 for the acceptor mouse after, with the mating of purebred CD-1 mouse, and the offspring who produces.Mother is a pure white, and the offspring is black.
Obtain the genetically engineered mouse of overexpression STPB-C
1, the structure of pFB-STPBC carrier
(1) preparation of target gene fragment
The pCMV-STPB-C plasmid with Hind III and BamH I double digestion after; 1% agarose gel electrophoresis is identified; And cutting-out contains the electrophoresis band of target gene fragment; From sepharose, reclaim purifying STPB-C encoding sox fragment with QIAEX-II Gel Extraction Kit, be resuspended in the TE solution (containing 10mmol/L Tris-HCl (pH=8.0) 6.057g in the 500ml TE solution) with 1mmol/L EDTA (pH=8.0) 1.8612g.
(2) preparation of carrier
Eukaryotic cell expression is carried the pFB-neo plasmid with HindIII and BamH I double digestion.Identify that through 1% agarose gel electrophoresis downcut the electrophoresis band that contains linear carrier, gel reclaims purifying, is resuspended in the TE solution.
(3) connection of recombinant plasmid
The target gene fragment that the enzyme switchback is received is connected (16 ℃ are spent the night) with linear pFB-neo plasmid with the T4 dna ligase.
(4) recombinant plasmid transformed intestinal bacteria
With recombinant plasmid and the unloaded plasmid difference of negative control transformed competence colibacillus intestinal bacteria Top10, be inoculated in the LB culture dish that contains penbritin (Amp) with the heat-shocked method, 37 ℃ of thermostat containers spend the night.Single bacterium colony on the picking plate is inoculated in the 4ml LB liquid nutrient medium that contains Amp at random, and 37 ℃ of joltings are spent the night.Carry out the screening of recon through PCR method, positive findings is delivered to order-checking, further analyzes and confirms.
2, the collection of recombinant virus particle
Get the EP pipe of a sterilization; Add 3 μ g pFB-STPBC recombinant plasmids, 3 μ g pVPack-GP (gag-pol-expressing vector) and 3 μ g pVPack-VSV-G (env-expressing vector), add the NaAc (3M) of 1ml absolute ethyl alcohol and 0.1 times of volume again.Behind the liquid-transfering gun mixing, place-30 ℃ of co-precipitation to spend the night.To mix plasmid in 4 ℃, 12, the centrifugal 10min of 000rpm, abandoning supernatant.Add 1ml 70% ethanol then, 12, the centrifugal 5min of 000rpm, abandoning supernatant.To mix plasmid and be stored in 4 ℃, spend the night.
Transfection 293T cell: a. observes 293T cell growing state in the Φ 60mm cell cultures plate, when cell grows to 80% plate, can be used for transfection experiment.B. remove old nutrient solution, add 4ml contain MBS [from the ViraPack Transfection kit (virus packing transfection reagent box) of Stratagene company) fresh medium, place 37 ℃ of incubators to cultivate.C. take out the centrifuge tube of include mixed plasmid; Add 450 μ l sterilization ultrapure water; Add 50 μ l Solution I and 500 μ l Solution II [Solution I and II are respectively from the ViraPack Transfection kit of Stratagene company (virus packing transfection reagent box)] again; Lightly behind the mixing, room temperature leaves standstill 10min with liquid-transfering gun.D. the 293T cell of take out cultivating joins aforesaid liquid in the petridish, gently rock plate, note that cell is not floating.Again new cell being put back to incubator cultivates.E.3h after, remove the liquid in the plate, add the nutrient solution that 4ml contains 25 μ M chloroquine.F.6h after, remove the liquid in the plate again, add the nutrient solution that 4ml does not contain chloroquine; Collect virion: collect culture supernatant, be filtered in the sterilization centrifuge tube, place liquid nitrogen immediately, and be placed on-70 ℃ of prolonged preservation with 0.45 μ m millipore filter.
Behind embodiment 1 said spermatogonial stem cells into mouse in-vitro separation cultural method cultivation stem spermatogonium 4d, STPB-C is crossed the expression vector virion infect this cell.The infection method is following: what method for preparing was taken out in (1) from-70 ℃ of cryogenic refrigerators contains STPB-C vector virus particle nutrient solution, places 37 ℃ of water-baths to dissolve.(2) dissolving back add rapidly DEAE (diethylammonium-2 hydroxy ethylamine) to final concentration be 10 μ g/ml.(3) remove nutrient solution old in the stem cell petridish, add the above-mentioned nutrient solution (the every hole of 24 well culture plates adds 200 μ l and contains the virion nutrient solution) that contains virion and DEAE.(4) add the fresh stem cell nutrient solution of 200 μ l to the every hole of 24 well culture plates behind the 3h.
1, the preparation of acceptor mouse
4-6 male mice in age in week is pressed 40mg/kg dosage abdominal injection busulfan (Busulfan, Sigma company), and after mixed fodder fed at least 4 weeks, the survival mice testicular spermatogenic function was destroyed, and carried out spermatogonium as the acceptor mouse and transplanted.
2, the stem spermatogonium cell suspension of pFB-STPBC virion infection is prepared
With 0.25% tryptic digestion culture, shift floating cell to centrifuge tube, 1, the 000rpm centrifugal collecting cell adds cold sterilization PBS damping fluid re-suspended cell, and the adjustment cell density is about 10
3/ μ l.
3, above-mentioned stem spermatogonium operation transplantation
Press 75mg/kg dosage, the abdominal injection Veronal sodium is anaesthetized male mouse.Clean the mouse health with 75% cotton ball soaked in alcohol, cut the abdominal cavity, extrude testis, and the cell suspension multiple spot is injected in the mouse convoluted seminiferous tubule, note avoiding injuring blood vessel with glass capillary along near the ventrimeson.After injection finishes, include testis in abdominal cavity, sew up the incision.Transplant after two months, male mouse of postoperative and female mouse are mated.
The result shows that stem spermatogonium transplants in 3 sterile male mice convoluted seminiferous tubules, and two obtain fertility, and common property is given birth to 46 offsprings, detects and Southern hybridization confirms that further wherein 2 is STPB-C transgenic positive offspring, sees Fig. 4,5 through PCR.Wherein Fig. 4 is the STPB-C transgenic positive mouse of PCR detection, M:DNA molecular weight standard; P: positive control; 2 and 7: the transgenic positive mouse; 1,3,4,5,6,8,9 and 10: wild-type mice.Fig. 5 is Southern hybridization checking STPB-C transgenic positive mouse; The M:DNA molecular weight standard; P: positive control; N: negative control; 1 and 3: the transgenic positive mouse; 2,4,5 and 6: wild-type mice.
Checking to the stem spermatogonium cultivated
1, the in-vitro transfection of stem spermatogonium
Carry out in-vitro separation and cultivate stem spermatogonium after about 4 days according to the method for the invention, the dna fragmentation of expressing goal gene or RNAi is passed through the virus vector transfection of spermatogonial stem cell.
2, the preparation of receptor
Receptor can be divided into congenital sterile with the day after tomorrow drug administration cause sterile two kinds.For the destruction of intact animal production of sperm ability, many uses is busulfan (Busulfan, busulfan) at present, and its mechanism of action is that busulfan can make the DNA alkylation, thereby destroys the propagation of acceptor stem spermatogonium.Thereby transplant through injection busulfan at least 4 Zhou Houke, the consumption of busulfan is confirmed according to the size of laboratory animal.
3, the transplanting of stem spermatogonium
The present invention adopts the convoluted seminiferous tubule injection to accomplish the transplanting of stem spermatogonium.Present method is to use the operating microscope injection device, and the clone that will pass through genetic modification is injected directly into the convoluted seminiferous tubule of receptor.Injection finishes, and sews up a wound, and the anti-inflammatory nurse guarantees surviving rate.The convoluted seminiferous tubule injection is fairly simple, easy master.After the transplanting,, can obtain generation mice through genetic modification through male mouse and female mouse mating after a while.
The result shows in 5 sterile male mice convoluted seminiferous tubules of stem spermatogonium gradation implantation of STPB-C gene silencing, 5 all fertilities, and common property is given birth to 82 generation mices.Through the PCR method screening and with its PCR product sequence verification, detect the positive mouse (see figure 6) of 5 STPB-C gene silencings.Fig. 6 shows that PCR detects STPB-C gene silencing mouse; The M:DNA molecular weight standard; P: positive control; 6,7,8,9 with 13:STPB-C gene silencing mouse; 1,2,3,4,5,10,11,12,14,15 and 16: wild-type mice.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; The present invention is not restricted to the described embodiments; That describes in the foregoing description and the specification sheets just explains principle of the present invention; The present invention also has various changes and modifications under the prerequisite that does not break away from spirit and scope of the invention, and these variations and improvement all fall in the scope of the invention that requires protection.The present invention requires protection domain to be defined by appending claims and equivalent thereof.
Claims (7)
1. the in-vitro separation cultural method of spermatogonial stem cells into mouse is characterized in that, comprises the steps:
Step 1 is collected mouse testis, adopts two step enzyme digestions to prepare cell suspension;
Step 2 utilizes the immunological magnetic bead sorting method to isolate stem spermatogonium;
Step 3, preparation stem spermatogonium nutrient solution carries out former being commissioned to train to stem spermatogonium and supports and the cultivation of going down to posterity.
2. the in-vitro separation cultural method of spermatogonial stem cells into mouse according to claim 1 is characterized in that, in the step 1, the magnetic bead that said immunological magnetic bead sorting method is used is the Thy1 magnetic bead.
3. the in-vitro separation cultural method of spermatogonial stem cells into mouse according to claim 2 is characterized in that, said Thy1 magnetic bead is CD90.
4. the in-vitro separation cultural method of spermatogonial stem cells into mouse according to claim 1 is characterized in that, in the step 3, adding STO cell feeder layer when cultivating is supported and gone down to posterity to said former being commissioned to train.
5. the employed substratum of in-vitro separation cultural method of spermatogonial stem cells into mouse as claimed in claim 1; It is characterized in that; Be made up of STO cell feeder layer and stem spermatogonium nutrient solution, said stem spermatogonium nutrient solution comprises MEM-α, FBS, L-glutaminate, LIF, bFGF, Regular Insulin, vitamins C, EGF, GDNF.
6. substratum according to claim 5 is characterized in that, said stem spermatogonium nutrient solution also comprises more than one in non-essential amino acid, beta-mercaptoethanol, the Sodium.alpha.-ketopropionate.
7. substratum according to claim 5 is characterized in that, the volumn concentration of said FBS is 8%-10%, and the content of said LIF is 10
2-10
4U/ml; The content of said bFGF is 0.02-0.03 μ g/ml, and the content of said IGF-1R activator is 0.04-0.06 μ g/ml, and the content of said EGF is 0.01-0.02 μ g/ml; Said ascorbic content is 40-60 μ g/ml, and the content of said GDNF is 0.01-0.02 μ g/ml.
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|---|---|---|---|
| CN2010102699313A CN102382799A (en) | 2010-08-31 | 2010-08-31 | In-vitro isolation and culture method of mouse spermatogonial stem cells and special culture medium thereof |
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Cited By (9)
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| CN103074297A (en) * | 2012-12-07 | 2013-05-01 | 内蒙古大学 | Livestock spermatogonial stem cell medium |
| CN103820383A (en) * | 2014-03-31 | 2014-05-28 | 广西大学 | Method of inducing spermatids with Ba-ma mini pig spermatogonia stem cells |
| CN104004708A (en) * | 2014-06-16 | 2014-08-27 | 内蒙古大学 | Method for separation and purification, long-term passage cultivation, cryopreservation and recovery of sheep spermatogonia stem cells |
| CN105969723A (en) * | 2016-06-01 | 2016-09-28 | 内蒙古大学 | Method for efficiently separating mouse spermatogonial stem cells |
| CN106085951A (en) * | 2016-06-23 | 2016-11-09 | 中国科学院昆明动物研究所 | A kind of method setting up the sustainable tree stem spermatogonium cell line passed on |
| CN106497869A (en) * | 2016-05-30 | 2017-03-15 | 南京医科大学 | A kind of stem spermatogonium cultivating system added without recombinant growth factors and application |
| CN109055306A (en) * | 2018-06-25 | 2018-12-21 | 佛山科学技术学院 | A kind of system and method for no feeder layer free serum culture spermatogonial stem cells into mouse |
| CN109456938A (en) * | 2018-11-16 | 2019-03-12 | 佛山科学技术学院 | A kind of method that spermatogonial stem cells into mouse is efficiently broken up to sperm in vitro |
| CN112961822A (en) * | 2021-02-25 | 2021-06-15 | 南方医科大学 | Testis organoid and construction method and application thereof |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103074297A (en) * | 2012-12-07 | 2013-05-01 | 内蒙古大学 | Livestock spermatogonial stem cell medium |
| CN103074297B (en) * | 2012-12-07 | 2014-07-30 | 内蒙古大学 | Livestock spermatogonial stem cell medium |
| CN103820383A (en) * | 2014-03-31 | 2014-05-28 | 广西大学 | Method of inducing spermatids with Ba-ma mini pig spermatogonia stem cells |
| CN104004708A (en) * | 2014-06-16 | 2014-08-27 | 内蒙古大学 | Method for separation and purification, long-term passage cultivation, cryopreservation and recovery of sheep spermatogonia stem cells |
| CN106497869A (en) * | 2016-05-30 | 2017-03-15 | 南京医科大学 | A kind of stem spermatogonium cultivating system added without recombinant growth factors and application |
| CN105969723A (en) * | 2016-06-01 | 2016-09-28 | 内蒙古大学 | Method for efficiently separating mouse spermatogonial stem cells |
| CN106085951A (en) * | 2016-06-23 | 2016-11-09 | 中国科学院昆明动物研究所 | A kind of method setting up the sustainable tree stem spermatogonium cell line passed on |
| CN106085951B (en) * | 2016-06-23 | 2020-01-14 | 中国科学院昆明动物研究所 | Method for establishing tree shrew spermatogonial stem cell line capable of being continuously passaged |
| CN109055306A (en) * | 2018-06-25 | 2018-12-21 | 佛山科学技术学院 | A kind of system and method for no feeder layer free serum culture spermatogonial stem cells into mouse |
| CN109456938A (en) * | 2018-11-16 | 2019-03-12 | 佛山科学技术学院 | A kind of method that spermatogonial stem cells into mouse is efficiently broken up to sperm in vitro |
| CN112961822A (en) * | 2021-02-25 | 2021-06-15 | 南方医科大学 | Testis organoid and construction method and application thereof |
| CN112961822B (en) * | 2021-02-25 | 2023-03-14 | 南方医科大学 | Testis organoid and construction method and application thereof |
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