**1.3 Needs for cryopreservation**

260 Current Frontiers in Cryobiology

quail *Coturnix japonica*, opossum *Monodelphis domestica* and marmoset *Callithrix jacchus*, etc. For example, sea lampreys are cyclostomes in a basal group of vertebrates. Comparative studies on lamprey and jawed fish, e.g., zebrafish, reveal key elements guiding jaw evolution. Characteristics shared between lamprey and other vertebrates but absent in nonvertebrate chordates include the presence of neural crest cells and a jaw. Comparative studies can direct us to the origins of these features. Therefore, study of these emerging model organisms does offer a unique approach to understand the relatedness of species in the living world. Another example illustrating the importance of new model systems in

studying the evolution of body plan is shown in Figure 1 (Kosik, 2009).

Fig. 1. The identification of miRNAs in different metazoan lineages revealed that the number of microRNAs (in brackets) is generally correlated with the complexity of body plan. Comparative studies involving "non-classical" model organisms further suggested a possible role of new microRNAs in evolutionary innovation. The figure is reproduced from

Kosik (2009) with permission from the publisher.

## **1.3.1 Archives of genetic resources**

Modern evolutionary developmental biology and molecular genetic studies on model organisms largely rely on manipulation of tissues and genomes. The advancement of technologies for genomic modification of these model organisms, in turn, affects the popularity of usage of particular model organisms. Eventually, this leads to a rapid increase in the number of transgenic/mutant strains in each popular species.

For the *Caenorhabditis* Genetic Center, mutants had been deposited by individual research groups and various genome wide mutagenesis projects such as the National BioResource Project for The Nematode and *Caenorhabditis elegans* Gene Knockout Consortium. In the National BioResource Project, random mutagenesis was performed for nematodes with UV in the presence of trimethylpsoralen. Affected genes are identified by screening with a genespecific primer set (Gengyo-Ando & Mitani, 2000). As of April 2010, about 4,400 mutants were available and mutants of some 2800 genes were being screened. Without convenient procedures for cryopreserving these species, maintenance of these strains is a heavy burden to these centers, and in other laboratories using these mutants extensively for their studies. On the other hand, passaging parasitic nematode models in plants and in donor animals, e.g., *Cooperia oncophora* in the cattle host (Borgsteede & Hendriks, 1979), is particularly labor intensive and costly and poses risk of cross contamination.

At Bloomington *Drosophila* Stock Center, more than 30,000 strains of *Drosophila* are currently present. Preparations are in progress to expand the facility to hold up to 70,000 stocks in order to meet the needs of stocking transgenic strains to be generated for a wide range of studies, including those made by tissue specific knocking-out of genes for the modeling of human diseases. In Flybase, 112,278 fly stocks were recorded for 2011 (Flybase FB2011\_07 Release Notes).

In The Jackson Laboratory, over 4,000 mouse strains have been deposited and are available to the public. At the Medical Research Council Harwell and at The European Mouse Mutant Archive, over 1300 and 2200 mouse strains are stocked respectively (Eppig & Strivens, 1999). In various mouse stock centers in Japan, including BioResources Center (Riken) and Trans Genic Inc., about 8,000 mouse strains are stocked. More than 16,000 of the 24,954 protein coding genes in the mouse genome have been modified by the International Knockout Mouse Consortium (IKMC), as conditional knockout alleles in embryonic stem cells. So far, more than 1,000 mutant mice, each containing one of these conditional knockout alleles, are made available to the community. The ultimate goal is to generate different targeted alleles in embryonic stem cells (for targeted mice generation) or in targeted mice, to be available to the research community worldwide (Skarnes et al., 2011). On the other hand, about 1,200 zebrafish lines and about 100 *Xenopus* lines have been archived in the Zebrafish International Resource Center and European *Xenopus* Resource Centers, respectively.

Since keeping live animals is costly in terms of requirements of space, consumables and manpower, strains not being used need to be cryopreserved to reduce the running cost. It is, therefore, a very important technology that keeps various genome-wide knockout consortia affordable to average research laboratories.

Cryopreservation of Embryos from Model Animals and Human 263

Fig. 2. Role of cryopreservation in assisted reproduction technology. Conflict between the time allowed for genetic diagnostic tests and the amount of genetic materials available (represented by the numbers of haploid genomes, N) from biopsies at different embryonic stages exists in a continuous pre-implantation genetic diagnosis program (dashed arrow). The release of the time constraint by a cryopreservation cycle after blastocyst biopsy is

The mammalian zygote (Figure 3D) is formed by fertilization of the oocyte by spermatozoa, normally in the oviduct. With advancement of the embryo culture technology, fertilization can be initiated outside the body (i.e. *in vitro* fertilization). The embryonic development continues up to hatched blastocysts, i.e. maximum of 4 days in mouse and 6 days in human, without compromising the development of the embryos after they are transferred into the recipient's uterus/oviduct. The preimplantation stag eembryo is composed of a single cell or multiple blastomeres surrounded by an outer membrane called the zona pellucida, glycoprotein layer of a thickness of about 6m in mouse and 8m in human. The embryos have an outer diameter of about 0.10 mm in mouse and 0.12 mm in human. The volume enclosed by the mouse zona pellucida is limited to about 200 ρl and the diameter of the

**2. Cryopreservation of mammalian preimplantation embryos** 

**2.1 Mammalian pre-implantation embryos at different stages** 

denoted by the solid arrow.
