Cryoconservation—archiving for the future
Peter H. Glenister, Claire E. Thornton
MRC Mammalian Genetics Unit, Harwell, Oxon OX11 ORD, UK
Received: 16 December 1999 / Accepted: 17 December 1999
Abstract. Mouse genetics is set to play a pivotal role in the key
post-genome challenge—the study of mammalian gene function.
Addressing this challenge will involve the development and ap-
plication of systematic mutagenesis approaches. The expanding
mouse mutant resource that will result threatens to overwhelm the
currently available animal facility space. Cryopreservation of both
mouse embryos and spermatozoa is currently widely employed for
the efficient archiving of mouse stocks. Distribution and dissemi-
nation of new and existing mouse strains is simplified by the
availability of extensive frozen archives. Also, the availability of
archives of frozen spermatozoa provides a potential powerful route
for the production of backcross progeny for rapid genetic mapping.
Moreover, frozen oocytes and ovaries may offer a valuable addi-
tion to the current cryopreservation approaches. Comprehensive
mouse mutant archives will provide an essential resource for mam-
malian genetics throughout the 21
It is generally accepted that we face a potential explosion of new
mouse models that threatens to swamp existing animal house space
and place an intolerable load on the infrastructural resources
needed to maintain these mice as conventional breeding colonies.
Several factors contribute to this burgeoning wealth of genetic
information. Firstly, despite the apparent size of the mouse mutant
resource, in fact mutations are known in only a small percentage
(maybe 1–2%) of the total number of mammalian genes. Several
institutions throughout the world are setting out to fill this “phe-
notype gap” (Brown and Peters 1996) by means of large system-
atic ENU (N-ethyl N-nitrosourea) mutagenesis programs (Brown
and Nolan 1998). Secondly, increasingly sophisticated ways of
manipulating the mouse genome have also resulted in a plethora of
transgenic, knock-out, and knock-in strains, all of which require
maintenance of some form so they are not lost to future research-
ers. Continuous breeding is costly and labor intensive, and there is
always the risk of loss owing to impaired reproductive perfor-
mance, death, or disease.
The creation of these new mutations and genetically manipu-
lated strains creates further burdens on animal facilities. Space is
required for studies such as inheritance testing and backcrosses for
genetic mapping. New transgenic and knockout lines need to be
expanded and bred to homozygosity. Most laboratories do not have
the resources to maintain all these new models as breeding colo-
nies, as well as carrying out fundamental research; therefore, al-
ternative strategies need to be considered.
Successful cryopreservation of mouse embryos was reported
independently by Whittingham et al. (1972) and Wilmut (1972).
The potential benefits of archiving mouse stocks for future re-
search were immediately recognized by geneticists (Whittingham
1974). Not only could stocks on which initial studies were com-
plete be economically preserved and removed from the shelf, but
critical mouse models could also be frozen and thus safeguarded
against disease, fire, and genetic contamination. Embryo banks
were established shortly afterwards at several major laboratories,
notably at the MRC Mammalian Genetics Unit (Harwell, UK), The
Jackson Laboratory, (Bar Harbor, Maine, USA), and the National
Institutes of Health, (Bethesda, Maryland, USA).
Several years later, in 1977, Whittingham published a success-
ful protocol for the cryopreservation of unfertilized mouse oocytes.
Cryopreservation of mouse ovarian tissue and recovery of fertile
mice following the grafting of frozen/thawed tissue had earlier
been reported by Parrott in 1958 and 1960. These investigations
showed that, although some live young were produced, the number
of surviving oocytes was low and the reproductive life of the host
females was shortened. Further progress on the freezing of mouse
ovaries and ovarian tissue remained limited until the 1990s.
Cryopreservation of spermatozoa from a wide variety of spe-
cies has been commonplace for many years. Indeed, pregnancies
and live-born offspring from cattle fertilized with frozen/thawed
semen were reported in 1952 (Polge and Lovelock 1952; Polge and
Rowson 1952). Today, the majority of cattle produced by artificial
insemination originate from frozen spermatozoa. Also in the 1950s
about 25 human births were achieved after insemination with fro-
zen human spermatozoa (Sherman 1979). Unfortunately, for rea-
sons that remain unclear, mouse spermatozoa proved far more
difficult to cryopreserve, and it is only during the 1990s that ap-
parently successful methods appeared in the literature (Tada et al.
1990; Okuyama et al. 1990; Takeshima et al. 1991; Nakagata
1994; Songsasen et al. 1997; Sztein et al. 1997; Marschall et al.
1999; Thornton et al. 1999).
Clearly, cryopreservation is now an invaluable tool for the
archiving of mouse genetic resources. All the preimplantation em-
bryonic stages of the mouse can be successfully frozen, from the
unfertilized oocyte to the blastocyst. Primordial follicles and ovar-
ian tissue can be frozen and are capable of producing fertilizable
eggs and liveborn offspring after transplantation to recipient fe-
males (Carroll and Gosden 1993; Candy et al. 1997; Gunasena et
al. 1997; Sztein et al. 1998). Now that it is possible to cryopreserve
mouse spermatozoa (from some strains, at least), exciting oppor-
tunities exist for the cryoconservation of the many thousands of
new mutations and genetically manipulated strains that will un-
doubtedly arise in the future. The advantages and disadvantages
involved in archiving various types of embryo, gametes, and tissue
Cryopreservation of Mouse Embryos
Since the enabling technology for mouse embryo freezing became
available in the 1970s, many laboratories have set up frozen em-
bryo banks for the secure and economical archiving both of pivotal
mouse lines and of those stocks which are little used at present but
may well be required in the future (see Appendix). The original
protocol described by Whittingham et al. in 1972 involved slow
cooling (<1°C/min) to −80°C with the embryos suspended in a
Correspondence to: P.H. Glenister; E-mail: P.Glenister@har.mrc.ac.uk
Mammalian Genome 11, 565–571 (2000).
© Springer-Verlag New York Inc. 2000