IN VITRO Volume13, No. 12, 1977 Allrightsreserved 9 ESTABLISHMENT OF DUCK CELL LINE DERIVED FROM EXPERIMENTAL TUMOR INDUCED BY 20-METHYLCHOLANTHRENE CHIL-YONG KANG AND JOHN A. SHADDUCK Department of Microbiology, The University of Texas Health Science Center, Southwestern Medical School. Dallas, Texas 75235 SUMMARY Experimental tumors developed in white Pekin ducks after intramuscular implantation of 20-methylcholanthrene. Cells derived from the primary tumor were adapted successfully to grow in vitro and have growth characteristics similar to that of established cell lines of mammalian origin. The cell density rises rapidly and the doubling time is approximately 19 hr. The duck cells have been cultured successfully for at least 80 passages in vitro. The continuously cultured cells have the characteristic chromosome pattern of duck, and the DNA of the duck cell line hybridized with duck liver DNA. We believe we have established a continuous cell line of avian origin. Electron-microscopic examinations of the tumor cells and RNA-directed DNA polymerase of the cell-free supernate show no evidence of endo- genous virus production. Key words: experimental tumors; ducks; 20-methylcholanthrene; cell line. INTRODUCTION Hundreds of continuous cell lines have been es- tablished from over 40 different species. How- ever, there is no known established cell line of avian origin which is capable of indefinite pro- liferation in culture. Marcovici and Prier (1) have derived a long-term culture from Pekin duck em- bryo which seems to have a limited generation time. Gey et al. (2} have been able to grow chicken fibroblasts continuously on a collagen substrate; however, these cells were unable to be grown on a glass surface for unlimited generations. The duck is ideal for the study of certain as- pects of avian leukosis-sarcoma virus {ALSV), since there are no endogenous sequences of ALSV in the duck cell DNA (3}. Accordingly, we were interested in establishing a cell line from a duck. Although the incidence of spontaneously occur- ring tumors in white Pekin duck is very low, a number of tumors, mostly of mesenchymal origin, have been reported in white Pekin ducks follow- ing the cutaneous application or intratracheal in- stillation of 20-methylcholanthrene IMCA) t4}. Fibromas, hemangiomas, neurofibromas, ganglioneuromas and a Pacinian corpuscle tumor resulted from local applications of MCA to skin 14}. Hemangiomas and gangliomas developed in the pectoral muscles of ducks which had received implants of MCA. Sarcomas also have been in- duced by Rous sarcoma virus in ducks (5}. We have induced experimental tumors in the muscle of white Pekin ducks by implanting 20-methyl- cholanthrene. The experimental tumors were sub- jected to culture in vitro, and the growth charac- teristics of cells have been studied. This report deals with the establishment of duck cell line de- rived from an experimental tumor induced by chemical carcinogen. This established duck cell line has growth characteristics similar to that of many known cell lines of mammalian origin. No endogenous RNA-directed DNA polymerase ac- tivity was found in the pelleted duck cell super- nate and no virus-like particle was observed in the cells. MATERIALS AND METHODS Inductions of tumors and cell growth in vitro. Six-week-old white Pekin ducks (Anas platy- rhynchos domesticus) were purchased from Trus- low Farm, Inc., Chestertown, Md. Fifty mg dry 20-methylcholanthrene {MCA) (Eastman Organic Chemicals} was implanted intramuscularly in the 849