The mammalian central nervous system (CNS) develops primarily during the embryonal period, but final structuring and tuning occurs postnatally. Before or soon after birth, the mammalian CNS loses its ability to fully regenerate after injury. Therefore, neuroregeneration studies are mostly conducted on rodent embryos (mice or rats) or derived in vitro preparations, such as primary cell or tissue cultures. However, an intrauterine surgical procedure is necessary to access rodent embryos, which usually means sacrificing the mother - an ethically questionable issue. Therefore, to study regenerative mechanisms of the CNS, we have used an alternative experimental animal: the grey South American short-tailed opossum (Monodelphis domestica), a marsupial without a pouch. Opossum neonates are very immature, and their CNS develops mostly postnatally, with the cortical plate not observed until postnatal day (P)3-5. Therefore, harming the mother is not necessary in order to investigate CNS development in opossums. Furthermore, opossums are unique among mammals in their ability to successfully regenerate the spinal cord after injury during the first two weeks of their life. After that period, the regenerative capability of their spinal tissue is lost. Thus, the first aim of this work was to characterize primary cortical cell cultures obtained from opossums at two developmentally different ages, P3-5 and P16-18, using immunofluorescent microscopy. The results showed a high percentage of neurons in primary cultures derived from both P3-5 and P16-18 opossums (>96% and >80% at DIV7, respectively). The second aim was to genetically silence activating transcription factor 3 (ATF3) in opossum primary cortical cultures, via transfection with specific siRNA molecules, to further investigate its role in neuroregeneration. The cell transfection protocol was optimized to reach the optimal transfection efficiency, and the ATF3 gene and protein levels were assessed with quantitative real-time PCR and Western blot, respectively. Altogether, the results indicate that primary cortical cultures obtained from opossum M. domestica are favorable preparation that can be used to investigate mechanisms underlying CNS regeneration, particularly the involvement of regeneration-related transcription factors, such as ATF3.