[A modified method of nuclear transfer for investigating early development of mouse embryos reconstructed with cumulus cell nuclei].

Objective: To establish a fast, simple, efficient and minimally invasive method for nuclear transfer (NT) to study the early development of mouse embryos reconstructed with cumulus cell nuclei in vitro.

Methods: With a sharp-tipped enucleation needle, an incision approximately 25 mm in length was made in the zona pellucida of a rat oocyte, through which the first polar body was removed and the metaphase II chromosome-spindle complex was gently aspirated along with a minimal volume of the cytoplasm by slightly pressing and vacuum aspiration of the oocyte. A cumulus cell nuclei from C57BL/6j mouse, 10-12 mm in diameter, was inserted into the perivitelline space of the enucleated oocyte.

[Effects of different electrofusion parameters on activation and early development of mouse embryos reconstructed by cumulus cell nuclear transfer].

Objective: To study the effects of different parameters for electrofusion on the activation and early development of mouse embryos reconstructed by cumulus cell nuclear transfer, and explore optimal parameters for electrofusion.

Methods: A C57BL/6j mouse cumulus cell nucleus 10-12 mm in diameter was inserted into the perivitelline space of an enucleated oocyte. The fusion of donor-recipient pairs was induced with different parameters for electrofusion (with variation in electric field intensity, pulse duration and pulse times).

Proteomics-based identification of proteins with altered expression induced by 12-O-tetradecanoylphorbol 13-acetate in nasopharyngeal carcinoma CNE2 cells.

Nasopharyngeal carcinoma (NPC) is a malignancy with high incidence in Southern China and South-East Asia. Etiology studies indicate that chemical carcinogen promoters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), are important factors causing NPC development. However, the mechanism of the TPA effect on NPC remains unclear.

[Analysis of protein expression maps of two metastatic colorectal carcinoma cell lines generated by two-dimensional gel electrophoresis].

Objective: To analyze the differentially expressed proteins in metastatic colorectal carcinoma and provide clues for the molecular mechanisms of colorectal cancer metastasis.

Methods: A pair of colorectal carcinoma cell lines SW620 and SW480 with different metastatic potentials from the same parent cell lines were studied. The protein expression maps of the two cell lines were obtained and optimized using two-dimensional gel electrophoresis (2-DE), and the differentially expressed protein spots associated with metastasis were analyzed by Melanie III software.

[Difference in gene expression profile of human polymeric immunoglobulin receptor-transfected mouse nasopharyngeal epithelial cells before and after EBV infection].

Objective: To study the gene expression profile of human polymeric immunoglobulin receptor gene (hpIgR)-transfected mouse nasopharyngeal epithelial cells transformed with n, n'-dinitrosoperazine (TMNE) before and after EBV infection using cDNA array and investigate the role of Epstein-Barr virus (EBV) infection in the tumorigenesis of nasopharyngeal carcinoma (NPC).

Methods: The total RNAs of hpIgR-transfected TMNE cells before and after EBV infection were extracted, reversely transcribed, and labeled with alpha -(32)P-dATP. The cDNA probes were hybridized to the Atlas mouse cancer array 1.

[Analysis of the difference in NIH3T3 cell protein expression profiles before and after TPA treatment using two-dimensional polyacrylamide gel electrophoresis and image analysis software].

Objective: To establish and optimize two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) for comparative analysis of the protein expression profiles in mouse fibroblast NIH3T3 cells before and after 12-O-tetradecanoy- lphorbol-13-acetate (TPA) treatment using image analysis software, so as to prepare for more intensive study in the identification of the proteins that mediate the biological effect of TPA.

Methods: The total cellular protein extracted from NIH3T3 cells with or without TPA treatment underwent 2-D PAGE and silver nitrate staining prior to the analysis of the differential protein expressions using image analysis software.

Results: Image analysis revealed obvious differential protein expressions of the cells in response to TPA treatment.

[Establishment of transgenic mouse models carrying human polymeric immunoglobulin receptor gene].

Objective: To establish transgenic mouse models carrying human polymeric immunoglobulin receptor (pIgR) gene to render the mice prone to EBV infection in normal condition, thereby to facilitate the study of the role of Epstein-Barr virus (EBV) in the pathogenesis of nasopharyngeal carcinoma.

Method: By means of microinjection, pIgR gene under the regulation by kerotinocyte-specific promoter ED-L2 was introduced into the pronuclei of mouse zygote, which was transferred into pseudo- pregnant female mice to induce nasopharynx-specific pIgR expression in the founder mice.

Result: Six out of the 16 founder mice (37.

Infection of immortalized human epithelial cell line Hacat with high-concentration Epstein-Barr virus.

Objective: To study the mechanism by which Epstein-Barr (EB) virus infects human epithelial cells.

Methods: Large-scale culture of marmoset lymphocytes B958 was carried out to extract and condense EB virus therein. Titrated by lymphocytes from fetal umbilical blood, the EB virus of high concentration was used to infect immortalized human epithelial cell line Hacat, followed by genomic DNA extraction from the Hacat cells and amplification of the special DNA sequence Bam HIw fragments of EBV genomic DNA by PCR.

[Construction of transgenic mice carrying enhanced green fluorescent protein gene by seminiferous tubule microinjection].

Objective: To study the feasibility of establishing transgenic mice carrying enhanced green flourescent protein (EGFP) gene by means of seminiferous tubule microinjection.

Methods: The vector expressing enhanced green fluorescent protein under the control of human cytomegalovirus immediate-early promoter (pCMV-EGFP) was selected and mixed with liposome in vitro. Microinjection at different doses of the liposome-entrapped plasmid DNA into the seminiferous tubules of male mice at different ages was performed to establish transgenic mice, which were made to mate with female mice at least 40 d after the microinjection.

Screening of single nucleotide polymorphisms in nasopharyngeal carcinoma associated genes by denaturing high-performance liquid chromatography.

Objective: To screen single nucleotide polymorphisms (SNPs) of 4 human genes at 6p21.3 by way of denaturing high-performance liquid chromatography (DHPLC).

Method: Four exons and 1 intron fragments in the PPP1R11, PPP1R10, FLOT1 and KIAA0170 genes at 6p21.

[Establishment of transgenic Xenopus laevis by intracytoplasmic sperm injection].

Objective: To study the feasibility of establishing transgenic laevis by intracytoplasmic sperm injection (ICSI).

Methods: The testes of mature Xenopus laevis were taken for the purification of their sperms, which was subsequently incubated with digitonin to prepare concentrate of the sperms. Treatment of the concentrate with linearized reporter vector pCMV-EGFP-N1 was performed, and the sperms were then injected into unfertilized ova harvested from female laevis, followed by culture and observation of the development of the ova.

Rapid isolation of cancer cells from tumor tissue by micromanuiplator and extraction of tiny amount of RNA.

Objective: To establish a rapid method for isolating and purifying cancer cells from tumor tissue and for RNA extraction from tiny amount of the cells thus obtained.

Methods: Frozen sections of the tumor tissues were prepared followed by rapid staining. Clusters of the cancer cells were isolated from the sections by micromanipulation technique and purified for extracting intact RNA that was subsequently assayed.