Conserving, Distributing and Managing Genetically Modified Mouse Lines by Sperm Cryopreservation - Genetic Engineering: Recent Developments in Applications - page 62

increase is achieved by collecting sperm from the vas deferens and epididymis
into a cryoprotective medium of 18% raffinose (w/v), 3% skim milk (w/v)
and 477 µM monothioglycerol. The sperm suspension is loaded into 0.25 mL
French straws and cooled at 37±1°C/min before being plunged and then stored
in LN2. Subsequent to storage, the sperm are warmed at 2,232±162°C/min
and incubated in in vitro fertilization media for an hour prior to the addi-
tion of oocyte cumulus masses from superovulated females. Sperm from 735
GM mouse lines on 12 common genetic backgrounds including C57BL/6J,
BALB/cJ, 129S1/SvImJ, FVB/NJ and NOD/ShiLtJ were cryopreserved and
recovered. C57BL/6J and BALB/cByJ fertilization rates, using frozen sperm,
were slightly reduced compared to rates involving fresh sperm; fertilization
rates using fresh or frozen sperm were equivalent in all other lines. Develop-
mental capacity of embryos produced using cryopreserved sperm was equiva-
lent, or superior to, cryopreserved IVF-derived embryos.
Conclusions/Significance
Combined, these results demonstrate the broad applicability of our approach
as an economical and efficient option for archiving and distributing mice.
introduction
Embryo cryopreservation is an effective strategy for managing mouse lines. Its
adoption has been limited by the cost, time and the number of animals required.
This is especially true for those lines where embryo yields are low, e.g. BALB/c.
Cryopreserving sperm is an attractive alternative. However, its widespread use has
been limited by the challenge of efficiently recovering cryopreserved sperm from
some commonly used inbred strains[1]. In our experience (Table 1) and in that of
others[2]-[5], the impaired fertility associated with cryopreserved mouse sperm is
dependent on genetic background, with sperm from the C57BL/6 backgrounds
being particularly sensitive. Yet, this strain is one of the most commonly used for
creating and maintaining genetically modified (GM) lines. More than 75% of the
670 mouse lines submitted to The Jackson Laboratory's Repository from January
2004 to January 2006 were maintained on a predominantly C57BL/6J back-
ground. Further, The National Institutes of Health are using C57BL/6 embryonic
stem (ES) cells to create a resource containing null mutations in every gene in
the mouse genome[6]. Thus, it is critical that an effective and efficient method of
cryopreserving and recovering C57BL/6 sperm be developed.
Since mouse sperm survive cryopreservation with reasonable success[2], the
key to an effective sperm cryopreservation and recovery scheme is maximizing
post-thaw fertilization capacity. In vivo, sperm develop the capacity to fertilize