Primary culture of cat intestinal epithelial cells in vitro and the cDNA library construction
Felids are the only definitive hosts of Toxoplasma gondii. To lay a foundation for screening the T. gondii-felids interaction factors, we have developed a reproducible primary culture method for cat intestinal epithelial cells (IECs). The primary IECs were isolated from a new born cat’s small intestine jejunum region without food ingress, and respectively in vitro cultured by tissue cultivation and combined digestion method with collagenase XI and dispase I, then purified by trypsinization. After identification, the ds cDNA of cat IECs was synthesized for constructing pGADT7 homogenization three-frame plasmid, and transformed into the yeast Y187 for generating the cDNA library. Our results indicated that cultivation of primary cat IECs relays on combined digestion to form polarized and confluent monolayers within 3 days with typical features of normal epithelial cells. The purified cells cultured by digestion method were identified to be nature intestinal epithelial cells using immunohistochemical analysis and were able to maintain viability for at least 15 passages. The homogenizable ds cDNA, which is synthesized from the total RNA extracted from our cultured IECs, distributed among 0.5–2.0 kb, and generated satisfying three-frame cDNA library with the capacity of 1.2 × 106 and the titer of 5.2 × 107 pfu/mL. Our results established an optimal method for the culturing and passage of cat IECs model in vitro, and laid a cDNA library foundation for the subsequent interaction factors screening by yeast two-hybrid.
KeywordsToxoplasma gondii cat intestinal epithelial cells (IECs) primary culture cDNA library
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- Alvarado-Esquivel, C., Torres-Castorena, A., Liesenfeld, O., García-López, C., Estrada-Martínez, S., Sifuentes-Álvarez, A., et al. 2009. Seroepidemiology of Toxoplasma gondii infection in pregnant women in rural Durango. Journal of Parasitology, 95, 271–274. DOI: 10.1645/GE-1829.1CrossRefGoogle Scholar
- Dubey, J.P., Beattie C.P. 2010. Toxoplasmosis of animals and humans. CRC Press, Second edition, pp. 313Google Scholar
- Grabinger, T., Luks, L., Kostadinova, F., Zimberlin, C., Medema, J.P., Leist, M., Brunner T. 2014. Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy. Cell Death & Disease, 5, e1228. DOI: 10.1038/cddis.2014.183CrossRefGoogle Scholar
- Zhan, K., Jiang, M., Sui, Y., Yan, K., Lin, M., Zhao G. 2017. Establishment of immortalized mouse intestinal epithelial cells line and study of effects of Arg-Arg on inflammatory response. In Vitro Cellular & Developmental Biology-animal, 53, 538–546. DOI: 10.1007/s11626-017-0143-4CrossRefGoogle Scholar