[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. The fusion of the donor-recipient pair was induced by electrofusion and nuclear formation was observed. The development of 2-cell, 4- to 8-cell and morula-stage embryos was observed after a 72-hour culture of the reconstructed oocytes in vitro. RESULTS: The modified NT method enabled one-step removal of the whole nucleus from the oocyte with confirmed reliability of complete nuclear removal by Hoechst 33342 staining of the removed nuclei examined under UV light. The process of enucleation took an average time of 15 s, and the survival rate of the enucleated oocytes reached 95%. The success rate of 76.7% was achieved for cumulus cell nucleus insertion into the zona pellucida of the enucleated oocytes and pronucleus formation occurred in 62.2% of the reconstructed oocytes with nuclear transfer. After 72 h of culture in vitro of the reconstructed oocytes in CZB medium, the percentage of embryos that developed into 2-cell, 4- to 8-cell and morula (more than 16 cells) stages were 57.5%, 39.1% and 27.6%, respectively. Microsatellite sequences (D7Mit22 and D4Mit204) were amplified from the DNA of the reconstructed embryos for identifying their origin, which was proved to be C57BL/6j mouse. CONCLUSION: The modified NT method is simple, minimally invasive, efficient and practicable to reconstruct mouse embryos with somatic cell nuclei.

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. In the present study, cells from a poorly differentiated squamous cell carcinoma NPC cell line, CNE2, were stimulated by TPA and proteomics technology was carried out to find protein discrepancies between control and TPA-treated cells. Results revealed that TPA treatment in CNE2 cells could upregulate the expression of "triosephosphate isomerase" and "14-3-3 protein sigma" and downregulate the expression of "reticulocalbin 1 precursor", "nucleophosmin", "mitochondrial matrix protein p1 precursor", and "stathmin". The changes in the expression of these genes suggested that TPA induced CNE2 cells to antiproliferation and to apoptosis, which was confirmed by subsequent apoptosis detection. Therefore, the effects of TPA on nasopharyngeal carcinoma cells were distinct from the effects on primary epithelial cells and we suggest reasons for these differences.

[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). Successful formation of the reconstructed embryos from donor-recipient pairs and the reconstructed embryos developing into the early embryonic stages (2-cell, 4-8-cell and morula stages) were observed and counted. RESULTS: The electric field intensity and pulse duration allowed variation during electrofusion within the range of 1 000-2 000 kV/cm and 40-160 ms, respectively. The donor-recipient pairs fused at very low rate when the parameters were below the allowed ranges, and disintegration or even death might occur when the parameters were above the ranges. Within these allowed ranges, variation of the electrofusion parameters did not produce significant impact on the ratio of the reconstructed embryos in 2-cell, 4-8-cell or morula stages (P<0.05). In addition, we suggested that pulse times be limited to 1-2. CONCLUSION: Optimal parameters for electrofusion are crucial for the fusion of donor-recipient pairs and the activation of the reconstructed embryo.

[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. RESULTS: The 2-DE expression maps of the two cell lines were well matched. The resolution of 2-DE was increased after using the nonlinear pH3-10 or overlapping narrow immobilized pH gradient strips, and 11 differentially expressed protein spots were identified after analysis using the software. CONCLUSION: Nonlinear pH3-10 and overlapping narrow immobilized pH gradient strips have higher separation efficiency for protein in 2-DE. The differentially expressed proteins separated will be useful for identifying metastasis-associated proteins of colorectal carcinoma and for establishing 2-DE database of the metastatic colorectal carcinoma cell lines.

[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 1.2, and the signals analyzed by AtlasImage software. RESULTS: Twenty-five genes differentially expressed in cells before and after EBV infection, including 23 up-regulated genes and 2 down-regulated genes. CONCLUSIONS: The gene expression profile of hpIgR-transfected TMNE cells may change after EBV infection, suggesting that these genes are probably involved in the tumorigenesis and progression of NPC.

[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. CONCLUSION: High-quality instruments for 2-D PAGE, skilled electrophoretic operation and efficient image processing are all essential in the reliable identification of stable functional proteins.

[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 (37.5%) was tested by PCR to be pIgR-positive, while Southern blotting identified only 2 positive mice. CONCLUSION: Transgenic mice harboring pIgR gene can be used potentially as a new model for studying the pothogenesis of nasopharyngeal carcinoma by EBV.

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. Southern blotting and in situ hybridization were employed to confirm the result of PCR. RESULTS: The results of PCR, Southern blotting and in situ hybridization all indicated that high-concentration EBV could infect Hacat cell line, but the rate of infection was rather low. CONCLUSION: There is an unknown pathway of infection for EBV entry into the epithelial cells other than the mediation by CR2 or pIgR.

[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. RESULTS: The condensed sperm we obtained were of high quality and after intracytoplasmic injection into the ova, a fertilization rate of 10% was achieved and 20% of the zygotes survived the neurula stages and developed into tadpoles, but all of which were slightly deformed. The integration ratio of green fluorescent protein (GFP) reporter gene was 81%, but GFP expression was not observed in the laevis. CONCLUSION: ICSI is a simple and practicable method for establishing transgenic Xenopus laevis.

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 6p21.3 isolated from the blood samples from 30 patients with nasopharyngeal carcinoma (NPC) and 28 healthy subjects were amplified by PCR, and the products analyzed by DHPLC. Some fragments of interest were sequenced and compared with the sequences available in National Center for Biotechnology Information (NCBI) database. RESULTS: In the 5 fragments, 4 new SNPs were identified and 4 known SNP loci and genotypes were confirmed. CONCLUSION: DHPLC is an effective, economical, and simple method with reliability for SNPs screening.

[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. Genomic DNA was extracted from the offspring of the founder mice for PCR and Southern blotting analysis, and the frozen sections of different tissues from 2 of the founders mice were prepared for fluorescence microscopic observation. RESULTS: Among the 41 mice receiving the microinjection, 32 survived and retained their mating ability and fertility, and among their 382 offspring 133 were positive for EGFP DNA as demonstrated by PCR, 15 of which were confirmed by Southern blotting analysis. The age of the mice or the doses of microinjection they received was not shown to impact the integration of EGFP gene, and fluorescence microscopy failed to detect significant EGFP expression in the tissues of the founder mice (P>0.05) in comparison with normal mice. CONCLUSION: Seminiferous tubule microinjection is simple and practicable to implement gene transfer in mice.

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. RESULTS: Clear vision was achieved by the staining of the sections. The cancer cell clusters were precisely isolated from which high-quality intact RNA was obtained as demonstrated by reverse transcriptase-PCR. CONCLUSION: Micromanuiplation can be effectively used in stead of laser capture microdissection to isolate and purify targeted cells from tiny amount of tissue samples, therefore making RNA extraction possible in this context.