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.

[Objective] To explore the variety of matrix metalloproteinase 9 (MMP9) and blood brain barrier (BBB) in cardiopulmonary resuscitation rats.[Methods] Eighty rats were randomly divided into 2 groups:the sham-operated group (n ＝ 40) and the resuscitation group (n ＝ 40).The two groups were anaesthetized and endotracheally intubated,the resuscitation group was also induced to cardiac arrest by aphysia.Then the rats were put to death and samples were taken at immediate,3 h,9 h,24 h,and 48 h.After that,the expression of MMP9,MMP9 mRNA,water content and Evans blue content in brain tissue were detected.Ultramicrostructure of brain tissue was observed with electron microscope.[Results] Compared to the sham-operated group,at 3 h,9 h,24 h and 48 h,the expression of MMP9 of resuscitation group was significantly changed.MMP9mRNA significantly increased.Water content statistically increased and so was Evans blue content.The change of ultramicrostructure in the resuscitation group at 3 h,9 h,24 h,and 48 h was obvious.[Conclusion] The expression of MMP9 and MMP9mRNA obviously increased in the cerebral ischemia model with CPR rats,and got to peak at 24 h.Water content and Evans blue content in brain tissue obviously increased in the cerebral ischemia model with CPR rats,BBB was destroyed,and the peak was 24 h.The injury of ultramicrostructure of brain tissue with electron microscope was obvious,and the peak was 24 h.

A mammalian nonclassical secretion sequence derived from mouse Engrailed2 homeoprotein (En2) was used to direct the secretion of the enhanced green fluorescent protein from Pichia pastoris. This signal peptide conferred the transport of enhanced green fluorescent protein into periplasm through an endoplasmic reticulum-golgi independent pathway, without inducing severe unfolded protein response as compared with Saccharomyces cerevisae α-factor preprosequence. This study implies that this mammalian nonclassical signal peptide could be developed as a useful tool for delivering cargoes to the cell surface of yeast.