The Surface Morphology and Fine Structure of CHO (Chinese Hamster Ovary) Cells Following Enucleation

Abstract

Chinese hamster ovary cells grown in monolayer culture and exposed to cytochalasin B were enucleated by centrifugation. Thereafter, the karyoplasts (the nucleated parts obtained from the bottoms of the centrifuge tubes) and the cytoplasts (the enucleated cytoplasmic parts attached to the coverslips) were allowed to recover and subsequently were examined by scanning and transmission electron microscopy. Cytochalasin B causes rapid and dramatic changes in the morphology of vertebrate cells growing in monolayer culture (1). In some instances the nucleus is segregated into an outpocketing on the free surface of the cell and may remain attached to the main body of the cell only by a thin stalk of cytoplasm. Occasionally the stalk breaks resulting in a separation of the cell into nuclear and cytoplasmic components (1-3). This process can be made to occur in virtually all cells in a culture by using centrifugal force during treatment with cytochalasin B so that the nucleus is pulled away from the cell (4, 5). Thus, it becomes possible to separate more than 95% of the cells in a culture into nuclear and cytoplasmic parts (6, 7). Both parts recover quickly from the effects of cytochalasin B. The nucleated part, called the karyoplast (8), consists of a nucleus enclosed in a thin shell of cytoplasm with an intact plasma membrane (9). The cytoplasmic part, called the cytoplast (8), contains the bulk of the cytoplasm and has representatives of all cytoplasmic organelles and systems including centrioles and microtubules. The karyoplasts continue to synthesize RNA and protein for at least several hours and remain intact for 48-72 hr. Cytoplasts synthesize protein for at least 12 hr and in this period can support metabolic activities of viruses (4, 6, 7). The cytoplasts survive for up to 48 hr.In this paper we describe observations by scanning and transmission electron microscopy on the structure and behavior of karyoplasts and cytoplasts derived from a Chinese hamster ovary (CHO) cell line. These observations extend those in earlier reports (8,9) and establish a basic background in the experimental use of karyoplasts and cytoplasts for the analysis of nuclear-cytoplasmic interactions. MATERIALS AND METHODSChinese hamster ovary cells were grown to semi-confluency on no. 1 thickness glass (round 18 mm) or plastic (25 mm) coverslips in Ham's F-12 medium containing 10% fetal calf serum. The plastic coverslips were punched out of the bottoms of Falcon plastic tissue culture dishes (Falcon Plastics, Oxnard, Calif.) with a heated piece of stainless steel tubing. The coverslips were then inserted cell-side down into centrifuge tubes containing 10 sg/ml of cytochalasin B (Imperial Chemical Industries, Ltd., Great Britain) in Ham's F-12 and centrifuged in a Sorvall SS-34 prewarmed head as previously described (4). Glass coverslips were spun for 40 min at 3000 to 3400 X g at 370, whereas the more sturdy plastic coverslips were spun at 17,000 X g for 20 min. After one centrifugation in cytochalasin B the enucleation was usually gr...

Conditioning of Karyoplasts for Producing Somatic Nuclear Transferred Gonadal Germ Cells in Domestic Chickens

“…Karyoplasts can contain ribosomes, occasional mitochondria and a few fragments of the endoplasmic reticulum [14], but lack centrioles, microtubules and Golgi apparatuses [15]. These characteristics need to be monitored carefully when karyoplasts are generated for the production of nuclear transferred offspring.…”

Observing fibrillar assemblies on scrapie-infected cells

“…1b-c and 4a). Minor differences in the cell surface appearance observed at 1 dpp may be attributed to variations in cell cycle stage, protein expression levels, and metabolism of individual cells [55,58]. After 2 dpp, most of the ScN2a cells exposed a slightly increased surface roughness (Fig.…”

Identification of key regulated events early in the life of hybrid animal cells constructed by nuclear transplantation.

“…By judicious choice of the parental cell types and the use of a variety of staining techniques, such reconstituted cells can be produced in large numbers and definitively identified immediately after fusion (8)(9)(10)(11). These cells, having a minimal contribution of cytoplasm from the nuclear donor cell type (12)(13)(14)(15), would appear to be excellent systems for investigating the interaction of cytoplasm and nucleus in determining a variety of cellular properties and events, including cell shape and cytoarchitecture and genome replication and expression. However, analyses of both reconstructed and cybrid cells performed so far have indicated that, except for a few instances (16)(17)(18)(19)(20)(21), exposure to a foreign cytoplasm has little long-term effect on a transplanted nucleus.…”