Preparation of Microcells For Use in Production of Transchromosomic Animals / 生物化学与生物物理进展

ABSTRACT

Microcell mediated chromosome transfer (MMCT) is a challenging technique for introducing exogenous chromosomes into interested mammalian cells. Combined with the somatic cell nuclear transfer technique, MMCT has been employed for producing transchromosomic animals of medical and agricultural value. Producing high quality of microcells is a key step in the success of MMCT. Eamamined by fluorescin staining and Giemsa staining, 0.2 mg/L colcemid was considered suitable for inducing high percentage of micronuclei in A9 (neo12) cells, without causing death of a mass of cells. Microcells were produced by centrifugation of micronucleated A9 (neo12) cells in Percoll density gradient containing 20 mg/L Cytochalasin B at 39 000 g. The resulting mixture of microcells, whole cells, karyoplasts and cytoplast fragments was filtered through 8 μm and 5 μm size membrane pores sequentially to obtain pure preparation of microcells. Microcells were then characterized by Giemsa staining and microcell PCR was first applied for examination of the quality of microcell preparation. The result showed that microcells containing our interest chromosomes-human chromosome 12 were equally distributed in the preparation, the preparation was suitable for use in generation of transchromosomic animals.