A Ujumqin sheep ear marginal tissue (USEM) fibroblast line, frozen in 147 cryovials with 4 x 10(6) cell each, was successfully established from 33 Ujumqin sheep ear marginal tissues using explant culture and cryopreservation techniques. The cells were morphologically consistent with fibroblasts. The growth curve was typical S-shape and the cell population passed through a lag phase, a logarithmic phase and a plateau phase. The population doubling time (PDT) was approximately 72 h. Tests for bacteria, fungi, viruses and mycoplasma were all negative. Isoenzyme polymorphism indicated that the genetic characteristics of the cell line were stable in vitro. Karyotyping analysis indicated that the chromosome number of a normal cell was of 2n = 54 and 95.4% of the entire population was diploid. The transfection efficiencies of six fluorescent proteins (pEGFP-N3, pEGFP-C1, pDsRed-N1, pEYFP-N1, pECFP-N1 and pECFP-mito) optimal at 48 h were from 18.5% to 30.1%. The cell line met all criteria from the American Type Culture Collection (ATCC). Not only has the germline of this important sheep breed been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somatic cloning research. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.

Both embryonic and adult neural stem cells (NSC) of rodents and humans have been isolated and cultured in vitro to date, and they are thought to have tremendous clinical promise in restoring the diseased or injured central nervous system. However, there are few counterpart reports on neural stem cells from birds. This study explored the isolation and culture system of duck neural stem and progenitor cells (NSPC) and investigated their major biological properties. Cells from the dorsal ventricular ridges of 10- to 13-d embryos were isolated, cultured, and purified by using a neurosphere assay. Growth kinetics and karyotype were analyzed. The differentiation potential of NSPC was detected by immunofluorescence. Apoptosis and acetylation level of histone 4 lysine 12 (H4K12) were assessed. Results indicated that the nestin-positive neurospheres derived from duck embryos were able to self-renew and differentiate into neurons, astrocytes, and oligodendrocytes, and were prone to be transfected with exogenous genes. Karyotype analysis showed that 95% (38 out of 40) of cells of the population were diploid. Apoptosis detection indicated that the apoptotic rate was elevated with increasing passage number and culture time. The cells were highly acetylated and exhibited typical NSPC properties. Efficiently transfected with fluorescent genes, they were available for gene therapy and suitable for research on intracellular distribution of proteins of interest.

Using tissue explantation and cryopreservation biotechniques, a Jingning chicken embryonic fibroblast bank was successfully established, which includes 43 embryo samples and a stock of 178 cryovials, each one containing 3.0×106 cells. Most of the cells were apparently fibroblasts in their morphology, and the population doubling time (PDT) was about 48 h. The total chromosome number of a diploid cell was 78. According to karyotyping and G-banding, the diploid rate in the cell bank was 97.62±2.12%. The cells were tested for microbial contamination and found free of infections from bacteria, fungi, viruses and mycoplasms. There was no cross-contamination from other cell lines as revealed by lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) isoenzyme polymorphisms. Six fluorescent proteins were transfected into the Jingning chicken embryonic fibroblasts, and the transfection efficiencies of these genes were between 10.1 and 41.9%. All the tests showed that the quality of the cell line conforms to the quality criteria of the ATCC (American type culture collection). This work succeeded not only in preserving the genetic resources of Jingning chicken, but it also established a new protocol to preserve endangered animal breeds.