CN101418281A - A kind of mouse sperm freezing protecting agent - Google Patents
A kind of mouse sperm freezing protecting agent Download PDFInfo
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- CN101418281A CN101418281A CNA2008102039373A CN200810203937A CN101418281A CN 101418281 A CN101418281 A CN 101418281A CN A2008102039373 A CNA2008102039373 A CN A2008102039373A CN 200810203937 A CN200810203937 A CN 200810203937A CN 101418281 A CN101418281 A CN 101418281A
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Abstract
The invention discloses a kind of C57BL/6J of being used for mouse and be the sperm freezing protecting agent of the genetically engineered mouse of genetic background with the C57BL/6J mouse; the component of described cryoprotectant comprises raffinose and skimmed milk; described component also comprises yolk, and the volume ratio of described yolk is 10～30%.Mouse sperm freezing protecting agent of the present invention; by having added yolk liquid; make the C57BL/6J mouse sperm and with the C57BL/6J mouse be the rate in vitro fertilization of genetically engineered mouse of genetic background than 4.5 times of the simple the highest raisings of cryoprotectant with raffinose and skimmed milk, effectively improved rate of fertilization.
Description
Technical field
The invention belongs to bioengineering field, particularly, is about a kind of mouse sperm freezing protecting agent, more specifically, is to be the sperm freezing protecting agent of the genetically engineered mouse of genetic background about a kind of C57BL/6J of being used for mouse and with the C57BL/6J mouse.
Background technology
The freezing preservation of seminal fluid has positive effect for animal experiment study, and influence freezing preservation effect key factor be employed cryoprotectant in process of cryopreservation first.
Among the freezing preservation to sperm, for the sperm freezing of other kinds, the mouse sperm freezing protection is the comparison difficulty, because the sperm of mouse has long and narrow acrosome and elongated tail.The mouse sperm acrosome is long and narrow, acrosome damage easily in labout process, and the empty pocket alveolation appears in acrosome, and makes sperm to be fertilized, and the damage of elongated tail in freezing recovery process makes motility of sperm descend, and be influential to rate of fertilization.
Present mouse sperm freezing protecting agent commonly used both at home and abroad generally adopts raffinose and skimmed milk to form, the raffinose of the general employing 18% of the ratio of raffinose and skimmed milk and 3% skimmed milk, and this refrigerant can be protected the sperm of most mouse species.But for the C57BL/6J mouse; this refrigerant but can not effectively be protected sperm; because the rate in vitro fertilization after the sperm recovery has only about 20%; though there is minority individuality sperm recovery back rate in vitro fertilization higher; but be fortuitous phenomena, just this refrigerant can not play a very good protection to the C57BL/6J sperm.
Simultaneously; because the C57BL/6J mouse is finished whole genome sequence in 2003; make it become in the mouse genetic background strain fully clearly; thereby become transgenic mice; the first-selected background mouse of genetically engineered mouse such as gene knockout mice; the preservation of the genetically engineered mouse that increase newly these every year; except the embryo preserves; also have sperm to preserve; and sperm saves as cost-effective preserving type; therefore need a kind ofly can be used for effectively protecting C57BL/6J mouse and C57BL/6J mouse is next its sperm of freezing preservation of sperm freezing protecting agent of the genetically engineered mouse of genetic background, thereby improves the anabiosis rate and the rate in vitro fertilization of its sperm.
Summary of the invention
Primary and foremost purpose of the present invention is to overcome existing mouse sperm freezing protecting agent cryoprotection C57BL/6J and be the sperm defective of the genetically engineered mouse of genetic background with the C57BL/6J mouse effectively, thereby a kind of new mouse sperm freezing protecting agent is provided.
Second purpose of the present invention is to provide the preparation method of described mouse sperm freezing protecting agent.
The 3rd purpose of the present invention is to provide the application of described mouse sperm freezing protecting agent.
According to the present invention; a kind of C57BL/6J of being used for mouse is provided and has been the sperm freezing protecting agent of the genetically engineered mouse of genetic background with the C57BL/6J mouse; the component of described cryoprotectant comprises raffinose and skimmed milk; described component also comprises yolk, and the volume ratio of described yolk is 10～30%.
According to mouse sperm freezing protecting agent of the present invention, preferably, the volume ratio of described yolk is 15～25%.
According to mouse sperm freezing protecting agent of the present invention, more preferably, the volume ratio of described yolk is 20%.
According to mouse sperm freezing protecting agent of the present invention, preferably, the volume ratio of described raffinose is 14.4%.
According to mouse sperm freezing protecting agent of the present invention, preferably, the volume ratio of described skimmed milk is 2.4%.
According to mouse sperm freezing protecting agent of the present invention, described with the C57BL/6J mouse be the genetically engineered mouse of genetic background comprise Y1F strain, Y3F strain, Y123F strain, Du+/-strain, Dukmga+/-mouse of strain, B6.CBA.DMT37 strain and TGIF strain.
The present invention also provides the preparation method of described sperm freezing protecting agent, may further comprise the steps:
A) skimmed milk and raffinose are dissolved in the tri-distilled water centrifuging and taking supernatant liquor successively;
B) get the SPF egg, take out yolk behind the derivation albumen, clean with the filter paper filter, puncture vitelline membrane with aseptic syringe needle, with the sucking-off of yolk liquid;
C) draw steps A in proportion) and B) gained composition, slowly stirred suspension to yolk dissolves, and 4 ℃ of centrifugal supernatant liquors are prepared into sperm freezing protecting agent.
The present invention also provides the application of described sperm freezing protecting agent, is used for the C57BL/6J mouse and is that the sperm freezing of the genetically engineered mouse of genetic background is preserved with the C57BL/6J mouse.
Mouse sperm freezing protecting agent of the present invention; by having added yolk liquid; make the C57BL/6J mouse sperm and with the C57BL/6J mouse be the rate in vitro fertilization of genetically engineered mouse of genetic background than 4.5 times of the simple the highest raisings of cryoprotectant with raffinose and skimmed milk, effectively improved rate of fertilization.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples only are used to the present invention is described but not are used to limit scope of the present invention.
R18S3 cryoprotectant among the present invention is meant that the 100ml tri-distilled water contains the mouse sperm freezing protecting agent of 18g raffinose and 3g skimmed milk.
SPF egg among the present invention (SPF is special pathogen free, i.e. specific pathogen free bacterium), the egg that the SPF chicken of raising available from Shanghai Slac Experimental Animal Co., Ltd. produces.
HTF nutrient solution among the present invention (being the HOF) prescription is as follows: NaCL101.6mM, KCL4.69mM, MgSO
4.7H
2O 0.2mM, KH
2PO
40.37mM, CaCL
2.2H
2O 2.04mM, NaHCO
325mM, glucose 2.78mM, Sodium.alpha.-hydroxypropionate 4.79mM, Sodium.alpha.-ketopropionate 0.33mM, penicillin 7.5mg/100ml, chain enzyme 5mg/100ml, bovine serum albumin 400mg/100ml, 0.5% phenol red (acid base indicator) be 20ul (ml).
In following examples, at first be equipped with the cryoprotectant R18S3 that does not contain yolk, adding volume ratio in this cryoprotectant respectively is 5%, 10%, 15%, 20%, 25%, 30% yolk is made R18S3+10% yolk, R18S3+15% yolk, R18S3+20% yolk, R18S3+25% yolk, the cryoprotectant of R18S3+30% yolk, with R18S3 and the cryoprotectant that respectively adds the yolk of different volumes ratio, be used for the C57BL/6J mouse and be genetically engineered mouse (the Y1F strain of gene knockout mice, Y3F strain, the Y123F strain of genetic background with the C57BL/6J mouse, Du+/-strain, and Dukmga+/-strain; With the B6CBA.DMT37 strain of transgenic mice, TGIF strain) the freezing preservation of sperm after, remove cryoprotectant, take out sperm, make it in vitro fertilization, calculate rate of fertilization at last.
In following examples, C57BL/6J male mice and provide the female mice of the C57BL/6J in ovocyte in vitro fertilization source to provide by Chinese Academy of Sciences's Shanghai Experimental Animal Center.The mouse of employed each strain, wherein Y1F, Y3F, Y123F, B6.CBA.DMT37, TGIF are provided by Chinese Academy of Sciences's Shanghai life science institute's nutrition and health research, Du+/-, Dukmga+/-provide by brain science institute of Shanghai life science institute of the Chinese Academy of Sciences.
Embodiment 1, cryoprotectant preparation
1.1, the configuration of R18S3 cryoprotectant: the skimmed milk (Becton of weighing 3g; Kickinson and Company); put into the 90ml tri-distilled water; dissolving is 2 hours in 60 ℃ of water-baths, after waiting milk powder particles dissolving fully, and the raffinose of weighing 18g (Sigma company product); put into wherein; add tri-distilled water to 100ml, continue in 60 ℃ of water-baths, to dissolve, dissolve fully up to the raffinose particle.With suspension under the condition of 15000g/min centrifugal 60 minutes, take out supernatant liquor, it is standby to put into clean glass bottle, and so the cryoprotectant of preparation contains the raffinose (volume ratio) of 2.4% skimmed milk and 14.4%.
1.2, the preparation of SPF yolk: in Bechtop, carry out following operation: get the SPF egg, at the end that air chamber is arranged of egg, the careful eggshell of knocking against hard is opened the egg film, the derivation that albumen is careful, until almost there not being albumen, yolk is placed on the large-scale filter paper then, blot clean albumen with filter paper, attention can not stave vitelline membrane, puncture vitelline membrane with sterilized syringe needle then,, put into clean glass cylinder (more than operate in Bechtop carry out) the sucking-off of yolk liquid.
1.3, the volume ratio according to 5%～30%, draw the yolk liquid and the above-mentioned R18S3 cryoprotectant of certain volume respectively, be configured to contain the cryoprotectant of 5%～30% yolk liquid; slow stirred suspension, treat the yolk dissolving after, under 4 ℃; 10000g/min centrifugal 30 minutes, gets supernatant liquor; continuation is under 4 ℃; 10000g/min centrifugal 30 minutes, gets supernatant liquor; after the packing, as the cryoprotectant of sperm.
Embodiment 2, cryoprotectant use
2.1; get the adult male mouse of listed each strain of table 1-table 8; put to death with the cervical vertebra dislocation method; belly with 75% alcohol disinfecting after; cut off skin and muscle; the careful testis of pulling out; expose epididymis; use the eye scissors separating out fat; tissues such as blood vessel and ligament, cut epididymis, the clean blood of cleaning on disinfectant filter paper; behind body fluid and the fat; put into each cryoprotectant of step 1.3 preparation of 120 μ l; cut off epididymis with scissors, treat that sperm flows out naturally after, can organize removal; soft sperm and the cryoprotectant thorough mixing of making, the straw of the 0.25ml that packs into then (available from French IMV company).
Wherein, pack into the method for straw of sperm suspension is as follows:
At first suck the HTF nutrient solution of about 100 μ l with syringe; Suck the air of 10mm then; Inhale the sperm suspensions about 10 μ l again; The last air of inhaling 10mm again, the straw two ends seal (filling HTF nutrient solution is because too gently swim in the surface of liquid nitrogen in order to prevent straw in the straw) with High-frequency machine.
2.2, after all straws all install, straw is placed in the precooling pipe, the precooling pipe is put into liquid nitrogen surface precooling, after 10 minutes, immerse in the liquid nitrogen stand-by rapidly.
2.3, the removal of cryoprotectant
When needing to use mouse sperm, need earlier cryoprotectant to be removed, method is as follows:
A) take out the required straw that thaws with tweezers in liquid nitrogen container, the sperm that moves into rapidly in 37 ℃ of constant temperature water baths thaws with in the container (centrifuge tube), it should be noted that this moment:
Guarantee 37 ℃ of constant temperature, one section bottom that places centrifuge tube of straw storage sperm;
Mouse sperm after freezing, to the adaptability of surrounding environment change a little less than, must careful operation.
B) after 15 minutes, thaw with taking out straw in the container (centrifuge tube), wipe the moisture of straw water with paper handkerchief from 37 ℃ sperms.
C) cut off straw sealing two ends place with straw, particularly: at first a HTF nutrient solution in the straw and a heat-sealing mouthful mid-way are cut off, the straw of cutting off the place is inserted the straw junctor (be the kapillary junctor actually, SIGMA company product, when buying SIGMA company kapillary, along with kapillary is given) the latex cap, regulate three-way control valve, again a seminal fluid in the straw and a heat-sealing mouthful mid-way are cut off, final operation straw junctor, take out sperm suspension, the sperm suspension in the straw after will thawing injects the good HTF solution of preheating, places 37 ℃, 5% CO
2Cultivated about 50 minutes in the incubator.
Embodiment 3, freezing recovery sperm in vitro fertilization
3.1, the collection of ovocyte
1) put to death the female mouse of the donor of handling through superovulation (8-12 age in week) in about 15-17 hours in injection hCG (the Ningbo second hormone factory produces for human chorionic gonadotrophin, hormone routinely) back;
2) mouse web portion is opened mouse peritoneal after with the alcohol swab wiping, with mobile abdominal visceral browsers such as tweezers, exposes uterus, uterine tube and ovary etc., takes out uterine tube, reduces blood, tissue juice and fat etc. as much as possible;
3) uterine tube is placed on the sterilising filtration paper, wipe bloodstain and tissue juice etc.;
4) uterine tube is placed the mineral oil of the culture dish that contains fertilization Solution H TF drop;
5) with one meticulous tweezers are fixed uterine tube, dissecting needle is punctured the oviducal portion of expanding, take ovocyte group out of to HTF cultivation drop with one.
3.2, the recovery sperm in vitro fertilization
1) recovery among the embodiment 2 is good sperm suspensions is from CO
2Take out in the incubator,, draw a certain amount of sperm, add in the HTF nutrient solution that contains ovum group of step 3.1 preparation, be placed on CO then with the edge of micropipet from sperm suspensions
2Fertilization moved on to ovum in the clean HTF drop with homemade kapillary after 4 hours in the incubator, put into CO again
2Overnight incubation in the incubator;
2) second day, calculate the ratio of two cells, calculate rate of fertilization, the result is shown in table 1-8.
Wherein: two cells=ovum sum-unusual ovum-unfertilized egg;
Rate of fertilization=(two cell count ÷ ovum sums) * 100%
Table 1
Table 2
Table 3
Table 4
Table 5
Table 6
Table 7
Table 8
By table 1-8 as can be known; after having added yolk liquid; C57BL/6J mouse sperm and be that the rate in vitro fertilization of the genetically engineered mouse of genetic background all has raising (the highest 4.5 times) in various degree than simple cryoprotectant with raffinose and skimmed milk with the C57BL/6J mouse, particularly:
The C57BL/6J mouse is after adding 15-25% yolk liquid, and rate of fertilization has improved 1.2-1.6 times;
Y1F strain mouse is after adding 15-30% yolk liquid, and rate of fertilization has improved 1.1-4.5 times;
Y3F strain mouse is after adding 10-20% yolk liquid, and rate of fertilization has improved 1.1-1.5 times;
Y123F strain mouse is after adding 20-25% yolk liquid, and rate of fertilization has improved 1.4-2.3 times;
B6.CBA.DMT37 strain mouse is after adding 15-30% yolk liquid, and rate of fertilization has improved 1.4-2.8 times;
TGIF strain mouse is after adding 15-30% yolk liquid, and rate of fertilization has improved 1.4-2.9 times;
Du+/-the strain mouse is after adding 10-30% yolk liquid, and rate of fertilization has improved 1.4-2.1 times;
Dukmga+/-the strain mouse is after adding 15-30% yolk liquid, and rate of fertilization has improved 1.0-1.8 times.
Further more as can be known, after having added 20% yolk liquid, the rate of fertilization of the mouse of each strain all reaches maximum, and the visible 20% yolk liquid that adds in the cryoprotectant that contains raffinose and skimmed milk is farthest improved the rate of fertilization of mouse.
Claims (9)
1, a kind of C57BL/6J of being used for mouse and be the sperm freezing protecting agent of the genetically engineered mouse of genetic background with the C57BL/6J mouse; the component of described cryoprotectant comprises raffinose and skimmed milk; it is characterized in that; described component also comprises yolk, and the volume ratio of described yolk is 10～30%.
2, sperm freezing protecting agent as claimed in claim 1 is characterized in that, the volume ratio of described yolk is 15～25%.
3, sperm freezing protecting agent as claimed in claim 2 is characterized in that, the volume ratio of described yolk is 20%.
4, sperm freezing protecting agent as claimed in claim 1 is characterized in that, the volume ratio of described raffinose is 14.4%.
5, sperm freezing protecting agent as claimed in claim 1 is characterized in that, the volume ratio of described skimmed milk is 2.4%.
6, sperm freezing protecting agent as claimed in claim 1; it is characterized in that, described with the C57BL/6J mouse be the genetically engineered mouse of genetic background comprise Y1F strain, Y3F strain, Y123F strain, Du+/-strain, Dukmga+/-mouse of strain, B6.CBA.DMT37 strain and TGIF strain.
7, a kind of preparation method of sperm freezing protecting agent according to claim 1 is characterized in that, may further comprise the steps:
A) skimmed milk and raffinose are dissolved in the tri-distilled water centrifuging and taking supernatant liquor successively;
B) get the SPF egg, take out yolk behind the derivation albumen, clean with the filter paper filter, puncture vitelline membrane with aseptic syringe needle, with the sucking-off of yolk liquid;
C) draw steps A in proportion) and B) gained composition, slowly stirred suspension to yolk dissolves, and 4 ℃ of centrifugal supernatant liquors are prepared into sperm freezing protecting agent.
8, a kind of application of sperm freezing protecting agent according to claim 1 is characterized in that, is used for the C57BL/6J mouse and is that the sperm freezing of the genetically engineered mouse of genetic background is preserved with the C57BL/6J mouse.
9, application as claimed in claim 8, it is characterized in that, described with the C57BL/6J mouse be the genetically engineered mouse of genetic background comprise Y1F strain, Y3F strain, Y123F strain, Du+/-strain, Dukmga+/-mouse of strain, B6.CBA.DMT37 strain and TGIF strain.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104720929A (en) * | 2015-03-16 | 2015-06-24 | 安徽农业大学 | Mouse early embryo acquisition device and method |
| CN106172368A (en) * | 2016-06-28 | 2016-12-07 | 华东师范大学 | A kind of mouse sperm frozen stock solution and application thereof |
| CN110074098A (en) * | 2019-06-18 | 2019-08-02 | 华东师范大学 | A kind of mouse sperm frozen stock solution and its preparation method and application |
-
2008
- 2008-12-03 CN CNA2008102039373A patent/CN101418281A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104720929A (en) * | 2015-03-16 | 2015-06-24 | 安徽农业大学 | Mouse early embryo acquisition device and method |
| CN104720929B (en) * | 2015-03-16 | 2016-10-05 | 安徽农业大学 | Mouse Embryos acquisition device and acquisition methods |
| CN106172368A (en) * | 2016-06-28 | 2016-12-07 | 华东师范大学 | A kind of mouse sperm frozen stock solution and application thereof |
| CN110074098A (en) * | 2019-06-18 | 2019-08-02 | 华东师范大学 | A kind of mouse sperm frozen stock solution and its preparation method and application |
| CN110074098B (en) * | 2019-06-18 | 2020-05-01 | 华东师范大学 | Mouse sperm cryopreservation liquid and preparation method and application thereof |
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Open date: 20090429 |