Stable transmission and transcription of newfoundland ocean pout type III fish antifreeze protein (AFP) gene in transgenic mice and hypothermic storage of transgenic ovary and testis

Abstract

Here we describe the generation of transgenic mice carrying type III fish antifreeze protein (AFP) gene and evaluate whether AFP type III protects transgenic mouse ovaries and testes from hypothermic storage. AFPs exist in many different organisms. In fish, AFPs protect the host from freezing at temperatures below the colligative freezing point by adsorbing to the surface of nucleating ice crystals and inhibiting their growth. The transgenic expression of AFP holds great promise for conferring freeze-resistant plant and animal species. AFP also exhibits a potential for the cryopreservation of tissues and cells. In this study, we have generated 42 founder mice harboring the Newfoundland ocean pout (OP5A) type III AFP transgene and established one transgenic line (the line #6). This study demonstrated that AFP gene construct has been stably transmitted to the mouse progeny in the F3 generations in the line #6. Furthermore, the presence of AFP transcripts was confirmed by RT-PCR analysis on cDNAs from liver, kidney, ovarian, and testis tissues of the mouse from F3 generation in this line. These results indicate that ocean pout type III AFP gene could be integrated and transmitted to the next generation and stably transcribed in transgenic mice. In histological analysis of testis and ovarian tissues of nontransgenic control and AFP transgenic mice it has been shown that both tissues of AFP transgenic mice were protected from hypothermic storage (+4 degrees C). The AFP III transgenic mice obtained for the first time in this study would be useful for investigating the biological functions of AFP in mammalian systems and also its potential role in cryopreservation.

Cryogenic effect of antifreeze protein on transgenic mouse ovaries and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries

“…Since there is no report on transgenic mammalian carrying this gene, it is difficult to find an explanation of how AFP works in mammalian system as in vivo under subzero condition. In our previous study, we generated transgenic mice carrying AFP gene and found AFP expression in the ovaries and testes (Bagis et al, 2006). Therefore, in this study AFP transgenic mouse was selected as a mammalian model.…”

“…The mice were fed ad libitum with autoclaved foodstuff and water. Transgenic mice carrying fish AFP type III gene were generated in our transgenic core facility (Bagis et al, 2006).…”

“…Transgenic synthesis of AFPs is a promising strategy for conferring freeze resistance to commercially important plant and animal species. It was clearly demonstrated in our previous study for the first time that type III AFP gene obtained from Newfoundland ocean pouts genome was effective to generate freeze-tolerant transgenic mouse lines when integrated and stably transmitted to the next generation in transgenic mammalian system (mice) (Bagis et al, 2006). The cryopreservation of ovarian tissue is a potentially useful technology for preservation of genetic resources of experimental, domestic, and wild animals.…”

Expression and partial characterization of an ice binding protein from a bacterium isolated at a depth of 3,519 meters in the Vostok ice core, Antarctica

“…Proteins that affect ice crystal structure and inhibit recrystallization can be exploited to improve the texture of ice cream (Regand and Goff, 2006), cold tolerance in plants (Wen-li et al, 2005), and the cryopreservation of mammalian tissues (Bagis et al, 2006). Currently, protein structural data for bacterial IBPs are restricted to a single genetic form (Garnham et al, 2011).…”

Antifreeze Peptides and Glycopeptides, and Their Derivatives: Potential Uses in Biotechnology

“…Several studies established transgenic plants: Type I AFP was introduced into tomato [163], potato [164], wheat [165], and tobacco [163,166], and type II AFP was introduced into tobacco [167]. Moreover, type III AFP-transplanted transgenic mouse ovaries were successfully cryopreserved by vitrification [168,169]. …”