CN108004204A - A kind of method of the high-quality egg mother cell of high flux screening - Google Patents
A kind of method of the high-quality egg mother cell of high flux screening Download PDFInfo
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- CN108004204A CN108004204A CN201810072343.7A CN201810072343A CN108004204A CN 108004204 A CN108004204 A CN 108004204A CN 201810072343 A CN201810072343 A CN 201810072343A CN 108004204 A CN108004204 A CN 108004204A
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Abstract
The present invention provides a kind of method of the high-quality egg mother cell of high flux screening, this method allows egg mother cell by ripe liquid I and II first, and culture environment reaches ripe in vitro, de- cumulus cell is carried out by de- ovarian cumulus solution again, primary election is carried out to the integrality of the membrane structure of egg mother cell at the same time, the semi-transparent incomplete egg mother cell of membrane structure is not in perivitelline, can eliminate a part of egg mother cell inferior in the process；The finally final election in isotonic no hormone solution, if the cell membrane of the egg mother cell of primary election cannot return to the state before de- ovarian cumulus solution treatment, then this egg mother cell must also be eliminated, and filter out high-quality egg mother cell so as to fast high-flux, the development for productivity cloning work.
Description
Technical field
The invention belongs to cell engineering field, passes through osmotic pressure treatment technology high throughput in particular to one kind
The method of screening high-quality egg mother cell.
Background technology
At present, in nuclear transfer research, the method that people screen egg mother cell used is substantially only as follows
Two kinds：The first is blind choosing, regardless of the egg mother cell of quality, is directly done；Second is to egg mother cell with brilliant cresyl blue
Dyeing, the egg mother cell for catching color are judged as that quality is preferable, and the egg mother cell for not catching color is judged as poor quality's.Document
Retrieval discloses：1.Theriogenology, 68 (2007) 1299-1304 authors：B.M.Manjunatha etc., is delivered《It is ripe
The preceding buffalo oocytes for having developmental potentiality with brilliant cresyl blue selection》Article content is that egg mother cell is placed in BCB dyeing liquors 90
Minute, compare selection rates of the various concentrations BCB to egg mother cell；Compare the yin and yang attribute egg mother cell diameter by selection
Difference；Different experiments group (control group, blank group, positive group, negative group) maturing rate is compared, blastocyst rate, as a result positive group is high
In other groups.2.Theriogenology, 63 (2005) 2194-2205, author：What H.Alm etc. was delivered《Use can be examined before maturation
Survey influence of the brilliant cresyl blue selection egg mother cell of G6PDH activity to the external blastocyst rate of ox》Article content be have detected through
Difference of the positive and negative egg mother cell of BCB dyeing in G6PDH activity is crossed, feminine gender group G6PDH activity is higher than positive group and experiment
Group.But positive group maturation in vitro rate, blastocyst rate are higher than other groups.3.Theriogenology, 57 (2002) 1397-1409, make
Person：What E.Rodriguez-Gonzalez etc. was delivered《Lamb goat oocytes are selected with brilliant cresyl blue》Article content is to study
Selection of the various concentrations BCB dyeing liquors to lamb goat oocytes, compares the in vitro fertilization of the egg mother cell after selection
Rate and maturing rate, it is found that the dyeing liquor of 26 μM of concentration is adapted to the selection of lamb goat oocytes.Positive ovum after selection is female
Cell is all significantly higher than negative egg mother cell in cell dia, development rate etc..4.Theriogenology, 61
(2004) 735-744, author：What MarcPujol etc. was delivered《Pass through small mother of brilliant cresyl blue (BCB) selection with developmental potentiality
Bovine oocyte》Article content is the choosing that have studied BCB dyeing liquors on the heifer egg mother cell for embryo production in vitro
Select effect.Developmental potentiality, egg mother cell diameter difference between positive egg mother cell and control group and mature cow are compared, is tied
Fruit shows：Different brackets egg mother cell selection rate is different, and positive egg mother cell diameter is more than negative egg mother cell, positive embryos capsule
Embryonic development rate is higher than negative group and control group, but is less than mature cow group.Domestic related Chinese Academy of Agricultural Sciences Beijing herding beast
What 8 people such as doctor research institute Qiao Limin delivered《Brilliant cresyl blue is dyed to bovine oocyte in vitro maturation and influence in vitro fertilization》
Brilliant cresyl blue dyeing liquor is used for screening of bovine oocyte etc..Obviously, second method needs to add dyestuff in the medium
And the culture of 90min is carried out, this method operation is complex, and screening efficiency is low, is unfavorable for mass production cloning work
Development.
The content of the invention
In view of there is the deficiency of technology, it is an object of the invention to provide a kind of high flux screening simple to operate is high-quality
The method of egg mother cell, to solve the problems, such as that current egg mother cell screening efficiency is low.
In order to achieve the object of the present invention, inventor combines scientific research experience for many years and persistent exploration and effort, finally
Different osmotic solution is used by sequential during embryo operation, making egg mother cell, culture environment is rapidly achieved into vitro
It is ripe, at the same using cell reaction of the pellicle to osmotic pressure come fast high-flux screening high-quality egg mother cell.Specifically, this hair
Bright technical solution is summarized as follows：
A kind of method of the high-quality egg mother cell of high flux screening, it is characterised in that this method comprises the following steps：
(1) cumulus oocyte complex selected is moved into ripe liquid I and cultivated, the composition of the ripe liquid I is：
TCM199+2.90~3.15mM glucose+0.85~0.95mM+0.52~0.60mML- of Sodium Pyruvate cysteines+90~
+ 9~11IU/mL of the liquor folliculi PMSG+9 of the streptomycin sulphate+9% of+90~110 μ g/mL of 110IU/mL penicillin~11%~
11IU/mLhCG；
(2) cumulus oocyte complex after step (1) is cultivated is moved into ripe liquid II and cultivated, the ripe liquid
II composition is：Half Guangs of TCM199+2.90~3.15mM glucose+0.85~0.95mM+0.52~0.60mM of Sodium Pyruvate L-
The liquor folliculi of the streptomycin sulphate+9% of+90~110 μ g/mL of propylhomoserin+90~110IU/mL penicillin~11%；
(3) cumulus oocyte complex for cultivating rapid (2) is moved into de- ovarian cumulus solution, concussion processing 0.5-2min,
Then the egg mother cell that perivitelline occurs in transfer carries out next step operation, and the de- ovarian cumulus solution composition is：PVA-TL-
+ 0.15~0.30mgBSA of Hepes+15~25mg/mL hyaluronidase+9~15mg mannitol；
(4) egg mother cell of rapid (2) primary election is moved into isotonic no hormone solution and washed, eliminating ooecium plasma membrane cannot recover
Egg mother cell, the composition of the isotonic no hormone solution is：TCM199+2.90~3.15mM glucose+0.85~
The sulfate chain of+90~110 μ g/mL of 0.95mM Sodium Pyruvate+0.52~0.60mML-+90~110IU/mL of cysteine penicillin
+ 7~9mg/mLHepes of liquor folliculi of mycin+19%~11%.
It is further preferred that the method for the high-quality egg mother cell of high flux screening as described above, wherein step (1) and step
(2) condition of culture is：5%CO2, saturated humidity, time 16-28h, 38-39 DEG C of temperature.
It is further preferred that the method for the high-quality egg mother cell of high flux screening as described above, wherein described in step (1)
The composition of ripe liquid I is：TCM199+3.05mM glucose+0.91mM Sodium Pyruvate+0.57mML- cysteines+100IU/mL
The liquor folliculi of streptomycin sulphate+10%+10IU/mLPMSG+10IU/mLhCG of the μ of penicillin+100 g/mL.
It is further preferred that the method for the high-quality egg mother cell of high flux screening as described above, wherein described in step (2)
The composition of ripe liquid II is：TCM199+3.05mM glucose+0.91mM Sodium Pyruvate+0.57mML- cysteines+100IU/mL
The liquor folliculi of streptomycin sulphate+10% of the μ of penicillin+100 g/mL.
It is further preferred that the method for the high-quality egg mother cell of high flux screening, wherein step (3) are selected after handling as described above
Go out the egg mother cell that perivitelline width is 4-6 μm and carry out next step operation.
It is further preferred that the method for the high-quality egg mother cell of high flux screening as described above, wherein described in step (3)
Taking off ovarian cumulus solution composition is：PVA-TL-Hepes+20mg/mL hyaluronidase+12mg mannitol+0.2mgBSA.
It is further preferred that the method for the high-quality egg mother cell of high flux screening as described above, wherein described in step (4)
The composition of isotonic no hormone solution is：TCM199+3.05mM glucose+0.91mM Sodium Pyruvate+0.57mML- cysteines+
The liquor folliculi of streptomycin sulphate+10%+7.5mg/mLHepes of+100 μ g/mL of 100IU/mL penicillin.
Compared with prior art, the present invention improves the maturation in vitro efficiency of egg mother cell by ripe liquid I and II first, makes
Culture environment is rapidly achieved maturation to egg mother cell in vitro, then carries out de- cumulus cell by de- ovarian cumulus solution, while female to ovum
The integrality of the membrane structure of cell carries out primary election, and the semi-transparent incomplete egg mother cell of membrane structure is not in perivitelline,
A part of egg mother cell inferior can be eliminated during this；The finally final election in isotonic no hormone solution, if the ovum of primary election is female
The cell membrane of cell cannot return to the state before de- ovarian cumulus solution treatment, then this egg mother cell must also be eliminated, so that
Can fast high-flux filter out high-quality egg mother cell, the development for productivity cloning work.
Embodiment
Following embodiments are only presently preferred embodiments of the present invention, not make limitation in any form to the present invention,
Any simple modification, equivalent change and modification that every technical spirit according to the present invention makees above example, still belongs to
In the protection domain of technical solution of the present invention.
Embodiment 1：The preparation of solution
The present invention judges the method and its solution formula of Oocyte quality quality by osmotic pressure processing porcine oocytes
Prescription method has sequential high sepage and isotonic solution totally five formulas, as follows respectively：
Ripe liquid I, is formulated and is：TCM199+3.05mM glucose+0.91mM Sodium Pyruvate+0.57mML- cysteines+
Liquor folliculi+10IU/mL the pregnant mare serum gonadotrop(h)in (PMSG)s of streptomycin sulphate+10% of+100 μ g/mL of 100IU/mL penicillin
(PMSG)+10IU/mL human chorionic gonadotrophins (hCG).
Ripe liquid II, is formulated and is：TCM199+3.05mM glucose+0.91mM Sodium Pyruvate+0.57mML- cysteines+
The liquor folliculi of streptomycin sulphate+10% of+100 μ g/mL of 100IU/mL penicillin.
De- ovarian cumulus solution, is formulated and is：PVA-TL-Hepes+20mg/mL hyaluronidase+12mg mannitol+0.2mgBSA.
Isotonic no hormone solution, is formulated and is：Half Guangs of TCM199+3.05mM glucose+0.91mM Sodium Pyruvates+0.57mML-
The liquor folliculi of streptomycin sulphate+10%+7.5mg/mLHepes of+100 μ g/mL of propylhomoserin+100IU/mL penicillin.
Hypertonic Hepes buffer solutions, are formulated and are：9.5gTCM199,0.05gNaHCO3, 0.75gHepes, 0.05g penicillin,
0.06g streptomysins, 1.755gNaCL, 3gBSA, add deionized water to be configured to 100mL.
Embodiment 2：The high flux screening of porcine oocytes
Test procedure is as follows：
(1) vacuum pump and its ancillary equipment are prepared, and it is 0.02 atmospheric pressure to adjust negative pressure value.Ovum is extracted with No. 16 syringe needles
Ovarian follicle of the diameter in 3~6mm on nest.Picking granular cell wraps up complete, the uniform cumulus oocyte of kytoplasm and answers under microscope
Fit (Cumulus-oocytecomplexes, COCs), the COCs selected is put into ripe liquid I and cultivates 24h, condition of culture
For 39 DEG C, 5%CO2, saturated humidity.Ripe liquid II is changed to after 24h, is then incubated for 16-24h.
(2) after cultivating 40h-48h, egg mother cell is moved into de- ovarian cumulus solution, 1min is handled in whirlpool concussion instrument, so
The egg mother cell for perivitelline occur with shifting ovum pin transfer afterwards carries out next step operation.If it should be noted that egg mother cell
Kytoplasm cannot be shunk, then illustrate that the membrane structure of oocyte membrane is imperfect, egg mother cell is dead；If the born of the same parents of egg mother cell
Matter is excessively shunk, then illustrates that the kytoplasm of egg mother cell is very few, poor quality；If after treatment, the kytoplasm of egg mother cell is received
Contracting, makes the width of the perivitelline between cell membrane and oolemma be in 5 μm or so, Oocyte quality at this time is optimal.
(3) the high-quality porcine oocytes preferably gone out are moved into isotonic no hormone solution and washed 3 times, eliminate ooecium plasma membrane not
The ovum that can recover.
(4) porcine oocytes after cytoplasmic membrane being recovered are changed in hypertonic Hepes buffer solutions after processing 30min, micro-
Microscopic observation.
Result of the test is as follows：
(1) not shrinkable egg mother cell is carried out lonely female activation, 1000 pieces of coprocessing, the spilting of an egg can be carried out without 1.
(2) egg mother cell that cannot recover elasticity is carried out lonely female activation, 1000 pieces of coprocessing.There is about 70% or so ovum
Mother cell can be with the spilting of an egg, but most of is unequal cleavage, and requires supplementation with and once electrically activate, and splitting time hysteresis
About 30~60min.
(3) egg mother cell that cannot recover elasticity is carried out stoning research, 1000 pieces of coprocessing.Have no idea to judge stoning
When remove kytoplasm number, very big difficulty is brought to operator.
(4) egg mother cell that cannot recover elasticity is placed in ripe liquid II, is observed after recovering 30min, still had again
30% ovum can recover elasticity, and residue 70% is too poor without the kytoplasm quality of the egg mother cell recovered.
(5) the rear egg mother cell for recovering elasticity is carried out clone's research, 100 pieces of coprocessing as nuclear receptor.Carrying out embryo
When tire is transplanted, the spilting of an egg is not seen.
(6) clone's research, 2000 pieces of coprocessing are carried out as nuclear receptor using picking out the preferable egg mother cell of quality.
When carrying out embryo transfer, more than the 93% reconstructed embryo spilting of an egg.
Claims (7)
- A kind of 1. method of the high-quality egg mother cell of high flux screening, it is characterised in that this method comprises the following steps：(1) cumulus oocyte complex selected is moved into ripe liquid I and cultivated, the composition of the ripe liquid I is： TCM199+2.90~3.15mM glucose+0.85~0.95mM+0.52~0.60mM of Sodium Pyruvate L-cysteines+90~ + 9~11IU/mL of the liquor folliculi PMSG+9 of the streptomycin sulphate+9% of+90~110 μ g/mL of 110IU/mL penicillin~11%~ 11IU/mL hCG；(2) cumulus oocyte complex after step (1) is cultivated is moved into ripe liquid II and cultivated, the ripe liquid II Form and be：TCM199+2.90~3.15mM glucose+0.85~0.95mM Sodium Pyruvate+0.52~0.60mM L-cysteines The liquor folliculi of the streptomycin sulphate+9% of+90~110 μ g/mL of+90~110IU/mL penicillin~11%；(3) cumulus oocyte complex for cultivating rapid (2) is moved into de- ovarian cumulus solution, concussion processing 0.5-2min, then The egg mother cell that perivitelline occurs in transfer carries out next step operation, and the de- ovarian cumulus solution composition is：PVA-TL-Hepes+15 ~25mg/mL hyaluronidase+9~15mg+0.15~0.30mg of mannitol BSA；(4) egg mother cell of rapid (2) primary election is moved into isotonic no hormone solution and washed, eliminate the irreclaimable ovum of ooecium plasma membrane Mother cell, the composition of the isotonic no hormone solution are：+ 0.85~0.95mM of TCM199+2.90~3.15mM glucose third The streptomycin sulphate of+90~110 μ g/mL of ketone acid sodium+0.52~0.60mM+90~110IU/mL of L-cysteine penicillin+ 19%~11%+7~9mg/mL of liquor folliculi Hepes.
- 2. the method for the high-quality egg mother cell of high flux screening according to claim 1, it is characterised in that step (1) and step (2) condition of culture is：5%CO2, saturated humidity, time 16-28h, 38-39 DEG C of temperature.
- 3. the method for the high-quality egg mother cell of high flux screening according to claim 1, it is characterised in that described in step (1) The composition of ripe liquid I be：TCM199+3.05mM glucose+0.91mM Sodium Pyruvate+0.57mM L-cysteines+100IU/ Liquor folliculi+10IU/mL the PMSG+10IU/mL of streptomycin sulphate+10% hCG of+100 μ g/mL of mL penicillin.
- 4. the method for the high-quality egg mother cell of high flux screening according to claim 1, it is characterised in that described in step (2) The composition of ripe liquid II be：TCM199+3.05mM glucose+0.91mM Sodium Pyruvate+0.57mM L-cysteines+ The liquor folliculi of streptomycin sulphate+10% of+100 μ g/mL of 100IU/mL penicillin.
- 5. the method for the high-quality egg mother cell of high flux screening according to claim 1, it is characterised in that after step (3) processing Select the egg mother cell that perivitelline width is 4-6 μm and carry out next step operation.
- 6. the method for the high-quality egg mother cell of high flux screening according to claim 1, it is characterised in that described in step (3) De- ovarian cumulus solution composition be：PVA-TL-Hepes+20mg/mL hyaluronidase+12mg mannitol+0.2mg BSA.
- 7. the method for the high-quality egg mother cell of high flux screening according to claim 1, it is characterised in that described in step (4) The composition of isotonic no hormone solution be：TCM199+3.05mM glucose+0.91mM Sodium Pyruvate+0.57mM L-cysteines Liquor folliculi+the 7.5mg/mL of streptomycin sulphate+10% Hepes of+100 μ g/mL of+100IU/mL penicillin.
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| CN109355254A (en) * | 2018-05-09 | 2019-02-19 | 南京优而生物科技发展有限公司 | It is a kind of to extract the Cell Buffer and its preparation method for collecting living cells |
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