Transgenesis in rabbit has provided numerous opportunities for livestock production. The development of nuclear transfer (NT) technology has improved the production of transgenic livestock by the combination of transgenic technology. However, the isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. 24-well cell culture plates were used to isolate cell lineages obtained from a single fibroblast clone transfected with the pRNAT-U6.1/Neo plasmid. Since single fibroblast clone does not grow well in fresh medium, the use of conditioned medium was evaluated. Meanwhile, the effect of initial cell density and linear-plasimid on transgenic fibroblast colony growth were investigated. The increasing initial cell density and lining plasmid could improve colony growth and expansion. There was a significant difference in the conditioned medium or initial cell density compared to the control group. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. When the transgenic fibroblasts were used as donor cells of nuclear transfer, the blastocyst rate were 23.5%. There was not a significant difference in the transgenic fibroblasts compared to the normal group. PCR or Multiplex-PCR assays were performed to detect the transfected fragment as well as autosomal β-actin DNA in single NT embryos. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production; further improvements in related technologies will facilitate the use of this method for the generation of the genetic engineering of rabbits.