3. Do not shake it to dissolve.
4. Solubilize the CaCl 2 .2H 2 O separately from the other reagents
before combining the solutions.
5. 1.0 mM L-glutathione reduced (GSH) must be added into
mHTF media before making the IVF drops.
6. Handle sperm gently to minimize agitation and pipette slowly.
7. Avoid any shaking or sudden temperature changes and do not
pipette. If the purpose is to conduct IVF, follow steps 1–3, but
resuspend the sperm pellet by gently adding TYH + MBCD
media and perform IVF as described by [19].
Acknowledgments
The authors acknowledge The University of Missouri Mutant
Mouse Resource and Research Center (NIH U42 OD01091820; http://www.mu-mmrrc.com).
References
1. Gordon JW, Scangos GA, Plotkin DJ, Barbosa
JA, Ruddle FH (1980) Genetic transformation
of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A
77:7380–7384
2. Palmiter RD, Brinster RL, Hammer RE,
Trumbauer ME, Rosenfeld MG, Birnberg
NC, Evans RM (1982) Dramatic growth of
mice that develop from eggs microinjected
with metallothionein-growth hormone fusion
genes. Nature 300:611–615
3. Robertson E, Bradley A, Kuehn M, Evans M
(1986) Germ-line transmission of genes
Fig. 5 A diagrammatic representation of post-thaw sperm dilution (a), centrifugation (b) and recovery
(c) procedures
Mouse Sperm Cryopreservation
411
4. Solubilize the CaCl 2 .2H 2 O separately from the other reagents
before combining the solutions.
5. 1.0 mM L-glutathione reduced (GSH) must be added into
mHTF media before making the IVF drops.
6. Handle sperm gently to minimize agitation and pipette slowly.
7. Avoid any shaking or sudden temperature changes and do not
pipette. If the purpose is to conduct IVF, follow steps 1–3, but
resuspend the sperm pellet by gently adding TYH + MBCD
media and perform IVF as described by [19].
Acknowledgments
The authors acknowledge The University of Missouri Mutant
Mouse Resource and Research Center (NIH U42 OD01091820; http://www.mu-mmrrc.com).
References
1. Gordon JW, Scangos GA, Plotkin DJ, Barbosa
JA, Ruddle FH (1980) Genetic transformation
of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A
77:7380–7384
2. Palmiter RD, Brinster RL, Hammer RE,
Trumbauer ME, Rosenfeld MG, Birnberg
NC, Evans RM (1982) Dramatic growth of
mice that develop from eggs microinjected
with metallothionein-growth hormone fusion
genes. Nature 300:611–615
3. Robertson E, Bradley A, Kuehn M, Evans M
(1986) Germ-line transmission of genes
Fig. 5 A diagrammatic representation of post-thaw sperm dilution (a), centrifugation (b) and recovery
(c) procedures
Mouse Sperm Cryopreservation
411