JAX provides cryopreserved mouse embryos from inbred and genetically modified mice to facilitate strain importation and microinjection experiments.
Reviewed 6/23/2004
The procedure used for freezing and thawing in The Jackson Laboratory Frozen
Embryo Repository is based on that described by Renard, J.P. and Babinet, C.
(1984). High survival of mouse embryos after rapid freezing and thawing inside
plastic straw with 1-2 propanediol as cryoprotectant. Journal
of Experimental Zoology 230, 443-448
Transfer the straw from the
liquid nitrogen storage freezer to a smaller container of liquid nitrogen
as quickly as possible.
Using forceps, hold the straw
near the label and hold in air for 40 seconds.
Place straw, label end up in
to the room temperature water bath (25 degrees Celsius) until the ice
disappears (~5 - 10 seconds).
Carefully wipe the straw dry
with a Kim Wipe.
Holding the straw firmly on
the embryo side of the PVA plug (yellowish color) cut through the middle
of the PVA plug.
Holding the straw near the
bottom seal, cut off the seal.
Using your index finger and
thumb, round off the end of the straw near the embryos.
Using the plunger, expel the
entire liquid contents of the straw as one drop into a 35mm Falcon dish.
Do not let the plug drop into the dish.
Wait 5 minutes. The embryos
will shrink considerably.
Transfer the embryos to 1ml
of M2. They will rapidly take up water and assume normal appearance.
Wash the embryos through 10 dishes of M2, using a new pipette for each wash (each dish should contain 1ml of media).
Your embryos should be transferred into your clean unit as soon as possible.
Reviewed
6/23/2004
The procedure used for freezing and thawing in The Jackson Laboratory Frozen
Embryo Repository is based on that described by Whittingham, et al.
[Whittingham, D., S. Leibo, et al. (1972). Survival of mouse embryos frozen to
-196°C and -269°C. Science.
178:411-14.]
Embryos require a slow thaw:
NaCl |
800.0 mg |
KCl |
20.0 |
KH2PO4 |
20.0 |
MgCl2.6H2O |
10.0 |
Dextrose |
100.0 |
Na2HPO4 |
115.0 |
Sodium pyruvate |
3.6 |
Penicillin G |
7.5 |
Streptomycin sulfate |
5.0 |
Add each chemical in order and mix carefully.
Gas for 5 minutes with 5% CO2, 5% O2 balanced with N2, using a 1 ml pipette
submerged in the media before adding BSA. Add BSA after gassing and then
incubate to dissolve. Filter with .22um filter and gas before refrigerating.
1000 ml | 200 ml | ||
---|---|---|---|
NaCl |
(FW 58.44, Sigma S-5886) |
5.9375 |
1.1875 |
KCl |
(FW 74.55,Sigma P-5405) |
0.3496 |
0.06992 |
MgSO4 7H20 |
(FW 246.5, Sigma M-1880) |
0.0492 |
0.00986 |
KH2PO4 |
(FW136.09, Sigma P5655) |
0.0504 |
0.01008 |
CaCl2 2H2O |
(FW 147, Sigma C-7902 |
0.3 |
0.06 |
(add directly) |
|
|
|
NaHCO3 |
(FW 84.01, Sigma S-5761) |
2.1 |
0.42 |
Glucose |
(FW180.16, Sigma G-6152) |
0.5 |
0.1 |
Na-pyruvate |
(FW110.0, Sigma P-4562) |
0.0365 |
0.0073 |
Na-lactate |
(FW 112.1, Sigma L-1375) |
3.42 ml |
0.683 ml |
Penicillin |
(FW 372.5, Sigma P-7794) |
0.075 |
0.015 |
Streptomycin |
(FW1457.4, Sigma S-9137) |
0.05 |
0.01 |
Phenolred (1%) |
(Sigma P-0290) |
0.20ml |
0.04 |
*BSA |
(Equitech-Bio BAC62-0050 |
4.0 |
0.8 |
Always gas the medium after every use.
Reference: Quinn P, Kerin JF and
Warnes GM (1985) Improved pregnancy rate in human in vitro fertilization
with the use of a medium based on the composition of human tubal fluid.
Fertility and Sterility 44: 493-498.
EDTA, disodium salt (ADD FIRST) |
(FW 372.2, Sigma) |
0.38 mg |
NaCl |
(FW 58.44, Fisher) |
559.5 mg |
KCl |
(Sigma) |
18.5 mg |
KH2PO4 |
(FW 136.09, Sigma) |
4.75 mg |
MgSO4.7H20 |
(Sigma) |
4.95 mg |
DL-Lactic Acid, sodium salt |
(MW 112.1, Sigma L-1375) |
0.174 ml |
D-Glucose |
(FW 180.16, Sigma G-6152) |
3.60 mg |
NaHCO3 |
(FW84.01, Sigma) |
210.0 mg |
L-Glutamine |
(MW 146.1, Sigma G-5763) |
14.5 mg |
Pyruvic Acid, sodium salt |
(FW 110.0, Sigma P-4562) |
2.2 mg |
Penicillin G |
(FW 372.5, Sigma P-7794) |
6.3 mg |
Streptomycin Sulfate |
(FW1457.4, Sigma S-9137) |
5.0 mg |
MEM Essential Amino Acids |
(GibcoBRL 11130-051)-10 ?l/ml |
1.0 ml |
MEM Non-essential AA |
(Sigma M-7145)-5 ?l/ml |
0.5 ml |
Lawitts, J.A. Biggers,
J.D. (1991). Optimization of mouse embryo culture media using simplex
methods. J Reprod Fert 91:543-556.
Lawitts, J.A. Biggers, J.D. (1992). Joint effects of sodium
chloride, glutamine, and glucose in mouse preimplantation embryo culture media.
Mol Reprod Dev 31:189-194.
Lawitts, J.A. Biggers, J.D. Culture of preimplantation embryos. In Methods
in Enzymology: Guide to Techniques in Mouse Development. P.M. Wasserman and
M.L. DePomphilis (eds.) 225:153-164. Academic Press. 1993
Erbach, G.T. Lawitts, J.A. Papaioannou, V.E. Biggers, J.D. (1994).Differential
growth of the mouse preimplantation embryo in chemically defined media.
Biol. Reprod. 50:1027-1033.
M2 medium is conveniently made from stock solutions.
Stock A
10X concentration (expires in 3 months)
Stock B 10X concentration (expires in 2 weeks)
Stock C 100X concentration (expires in 2 weeks)
Stock D100X concentration (expires in 3 months)
Stock E 100X concentration (expires in 3 months)
Prepare stocks A, B, C, and D
Prepare stock E
per 200 ml |
---|
Stock A(x10), 20 ml |
B(x10), 3.2 ml |
C(x100), 2 ml |
D(x100), 2 ml |
E(x10), 16.8 |
Distilled H2O, 156 ml |
BSA, 0.8 g |
*Store stock solutions and M2 in the refrigerator at 4 degrees C.
Used as an osmotic buffer for embryo cryopreservation.
Sucrose Sigma Cat. # S-1888
Component | Sigma cat. # | Size ordered | g/200 mL |
---|---|---|---|
NaCl |
S-5886 |
500g |
11.068 |
KCl |
P-5405 |
250g |
0.712 |
KH2PO4 |
P-5655 |
100g |
0.324 |
MgSO4*7H2O |
M-1880 |
500g |
0.586 |
soduim lactate |
L-7900 |
100g |
8.698 |
glucose |
G-6152 |
100g |
2 |
penicillin |
P-4687 |
10,000,000 u |
0.12 |
streptomycin |
S-1277 |
5g |
0.1 |
Component |
g/20 mL | ||
---|---|---|---|
NaHCO3 |
S-5761 |
500g |
0.4202 |
Phenol red |
P-5530 |
5g |
0.002 |
Component | g/10 mL | ||
---|---|---|---|
sodium pyruvate |
P-4562 |
25g |
0.036 |
Component | g/50 mL | ||
---|---|---|---|
CaCl2*2H2O |
C-7902 |
500g |
1.26 |
Component | g/250 mL | ||
---|---|---|---|
HEPES |
H-6147 |
100g |
14.895 |
Phenol red |
0.025 |
||
Albumin, bovine |
A-2153 |
50g |
0.8 |
|
Fisher Cat.# | ||
---|---|---|---|
150 ml filter system |
SCGP U01 RE |
12 pack |
.22 micrometers |
250 ml filter system |
SCGP U02 RE |
12 pack |
.22 micrometers |
Component | Sigma cat. # |
---|---|
1,2-Propanediol |
P-1009 |
Store at 4°C, discard after 1 week.
10X EBSS (see below) |
20 ml |
E. Amino Acid 50X |
4 ml |
100X Vitamins |
2 ml |
Na Pyruvate |
5 mg |
K Penn |
15 mg |
Strep |
10 mg |
L-Glutamine |
58.4 mg |
NaHCO3 |
0.44 g |
EDTA |
0.76 mg |
200 ml
CaCl2 2 H2O |
(Sigma C-7902) |
0.53 g |
KCl |
(Sigma) |
0.80 g |
MgSO4 7 H2O |
(Sigma) |
0.40 g |
NaCl |
(Fisher) |
13.60 g |
NaH2PO4 |
(Sigma S-5011) |
0.25 g |
D-Glucose |
(Sigma G-6152) |
2.0 g |
Calcium dihydrate should be dissolved first in a separate container with
millipore H2O .
**Add the CaCl2 last after all the other chemicals have dissolved.
2, 2, 2 Tribromoethanol
Tertiary amyl alcohol
25 g Tribromoethanol in 15.5 ml
amyl alcohol.
Store in dark (wrap bottle in foil).
1 ml of stock solution to 50 ml of
isotonic saline.
Warm to 40ºC, shake well, then store in refrigerator.
0.015 ml per gram body weight or 0.4 ml per average adult mouse.
(D+) raffinose pentahydrate (Sigma R-0250) |
18% |
Skim Milk Dehydrated (Difco* 0032-17-3) |
3% |
Culture grade water |
|
*(Difco has been bought by Becton-Dickinson)
Here are two recipes for making the cryoprotectant (CPA). They differ in how
clear the solution will be before filtering; we can detect no difference in
function.
Notes
Reference: Nakagata, N. (1996). Use of cryopreservation techniques of embryos and spermatozoa for production of transgenic (Tg) mice and for maintenance of Tg mouse lines. Lab-Anim-Sci 46(2): 236-8.
PMSG (pregnant mare serum
gonadotropin) is available from:
Sigma Chemical Company
P.O. Box 14508
St. Louis, MO 63178
It is available in 10,000 I.U. vials Cat. #G-4877. Working solutions are
made up by adding 400 ml of isotonic saline to a 10,000 I.U. vial of powdered
hormone, which is then stirred with a magnetic stirrer for 5-6 minutes. This
solution can then be packaged into 12 x 75 mm disposable plastic tubes with
caps (we use BIORAD tubes as these are available in colors for coding purposes
- PMS and HCG can be kept in different color tubes to avoid mix up) and frozen
until needed. Frozen preparations are not kept for longer than three months.
HCG (human chorionic
gonadotropin) is also available from Sigma
Sigma Chemical Company
P.O. Box 14508
St. Louis, MO 63178
It is also available in 10,000 I.U. vials Cat. #CG-10. It is diluted in the
same way as PMS described above.
For a working solution inject the 10 ml of diluent supplied into the vial of
powdered hormone (10,000 I.U.) with a syringe and needle, withdraw the mixed
solution and add to 390 ml of isotonic saline. This solution can then be
packaged into 12 x 75 mm disposable plastic tubes with caps as for the PMS, but
in different color tubes if possible.
These dilutions will yield 2.5 I.U. of
hormone per 0.1 ml.
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