ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2016, Vol. 47 ›› Issue (6): 1272-1279.doi: 10.11843/j.issn.0366-6964.2016.06.025

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Isolation,Culture and Identification of Rabbit Embryonic Fibroblasts

WEI Ru-xue,ZHAO Xue-ming,HAO Hai-sheng,DU Wei-hua,ZHU Hua-bin*   

  1. (Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 100193,China)
  • Received:2015-12-14 Online:2016-06-23 Published:2016-06-23

Abstract:

This research was conducted to establish the stable rabbit embryonic fibroblast cell lines and identification methods. Rabbit embryonic fibroblasts were isolated from 14-15 d rabbit fetus by trypsin enzyme digesting skin tissues, and passaged in vitro steadily. Morphology observation, growth curve measurement, immunofluorescence and karyotype analysis were applied to cell lines identification. The sex of 2 cell lines were determined by amplifying core sequence of rabbit SRY gene. The results showed that rabbit embryonic were spindle shaped and fibroblasts grew fast in vitro, conforming to the pattern of  “latent phase-logarithmic growth phase-stagnate phase”. Immunofluorescence assay showed that vimentin and fibronectin staining were positive, while cytokeratin staining was negative, indicated the high purity and activity of rabbit embryonic fibroblast cells. The result of chromosome analysis showed that 80% ploidy of one cell lines maintained normal (2n=44) when cultured up to passage 5. For cell lines sexing, SRY-PCR analysis suggested the 2 determined cell lines were male. These results indicated that our research could be used to establish rabbit embryonic fibroblast cell lines and determine sex, providing adequate and reliable fibroblast cells for transgenesis, cloning and iPS rabbit research.

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