Biomedical subjects

Fanyi Zeng

Publications and source records attributed to Fanyi Zeng.

9 recordsLinked to original sources

PubMed2006

Basonuclin: a novel mammalian maternal-effect gene.

Basonuclin is a zinc-finger protein found in abundance in oocytes. It qualifies as a maternal-effect gene because the source of pre-implantation embryonic basonuclin is maternal. Using a transgenic-RNAi approach, we knocked down basonuclin specifically in mouse oocytes, which led to female sub-fertility. Basonuclin deficiency in oocytes perturbed both RNA polymerase I- and II-mediated transcription, and oocyte morphology was affected (as evidenced by cytoplasmic and cell surface abnormalities). Some of the affected oocytes, however, could still mature to and arrest at metaphase II, and be ovulated. Nevertheless, fertilized basonuclin-deficient eggs failed to develop beyond the two-cell stage, and this pre-implantation failure accounted for the sub-fertility phenotype. These results suggest that basonuclin is a new member of the mammalian maternal-effect genes and, interestingly, differs from the previously reported mammalian maternal-effect genes in that it also apparently perturbs oogenesis.

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PubMed2005

Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes.

RNA interference (RNAi) is a conserved eukaryotic mechanism by which double-stranded RNA (dsRNA) triggers the sequence-specific degradation of homologous mRNAs. Recent concerns have arisen in mammalian systems about off-target effects of RNAi, as well as an interferon response. Most mammalian cells respond to long dsRNAs by inducing an antiviral response mediated by interferon that leads to general inhibition of protein synthesis and nonspecific degradation of mRNAs. Moreover, recent reports demonstrate that under certain conditions, short interfering RNAs (siRNAs, 21-25 bp) may activate the interferon system. Mouse oocytes and preimplantation embryos apparently lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In the present study, we analyzed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes expressing long dsRNA and find no evidence of off-targeting. We also report that genes involved in the interferon response pathway are not expressed in mouse oocytes, even after exposure for an extended period of time to long dsRNA.

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PubMed2005

RNA transcript profiling during zygotic gene activation in the preimplantation mouse embryo.

Zygotic gene activation is essential for development beyond the 2-cell stage in the preimplantation mouse embryo. Based on alpha-amanitin-sensitive BrUTP incorporation, transcription initiates in the 1-cell embryo and a major reprogramming of gene expression driven by newly expressed genes is prominently observed during the 2-cell stage. Superimposed on genome activation is the development of a transcriptionally repressive state that is mediated at the level of chromatin structure. The identity of the genes that are expressed during the 1- and 2-cell stages, however, is poorly described, as are those genes involved in mediating the transcriptionally repressive state. Using the Affymetrix MOE430 mouse GeneChip set, we characterized the set of alpha-amanitin-sensitive genes expressed during the 1- and 2-cell stages, and we used Expression Analysis Systematic Explorer (EASE) and Ingenuity Pathway Analysis (IPA) to identify biological and molecular processes represented by these genes, as well as interactions among them. We find that although the 1-cell embryo is transcriptionally active, we did not detect any transcripts present on the MOE430 GeneChip set to be alpha-amanitin-sensitive. Thus, what the BrUTP incorporation represents remains elusive. About 17% of genes expressed in the 2-cell embryo are alpha-amanitin-sensitive. EASE analysis reveals that genes involved in ribosome biogenesis and assembly, protein synthesis, RNA metabolism and transcription are over-represented, suggesting that genome activation during 2-cell stage may not be as global and promiscuous as previously proposed. IPA implicated Myc and Hdac1 as candidate genes involved in genome activation and the development of the transcriptionally repressive state, respectively.

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PubMed2005

In utero transplantation of human hematopoetic stem cells into fetal goats under B-type ultrasonographic scan: an experimental model for the study of potential prenatal therapy.

OBJECTIVE: Using fetal goats as animal models, to establish the methodology of in utero transplantation of human hematopoeitic stem cell (HSC) under B-scan ultrasonographic guidance for prenatal therapy. STUDY DESIGN: Human HSC were directly injected into the peritoneal cavities of the recipient fetal goats at 45-55 days of gestation (term: 145 days) under the guidance of B-type ultrasound scan. After birth, the peripheral blood was collected for fluorescence assisted cell sorting (FACS), quantitative real-time PCR and fluorescence in situ hybridization (FISH) to detect and analyze the presence of human cells in the recipients. RESULTS: The 32 recipients were born alive except one miscarriage. To test for the presence of human-goat chimeras, cells from 13 randomly selected transplanted goats were collected. FACS analyses showed the presence of human cells in all the transplanted goats tested. The average proportion of CD34+ cells and GPA+(glycophorin A) cells in the peripheral blood were 1.34 +/- 1.10% and 2.80 +/- 2.10%, respectively. No CD34+ or GPA+ cells were found in the non-transplanted goats tested. The results of the quantitative real-time PCR in three engraftment goats were 1.2 x 10(4), 2.9 x 10(4), and 3.2 x 10(4) copies of human GPA DNA per mug of genomic DNA. FISH experiments showed that cells containing human specific alpha-satellite DNA sequence were present in the peripheral blood of the transplanted goats. CONCLUSIONS: The method described herein is safe and reliable, with low miscarriage risk and high chimerism rate. This approach may provide a promising animal model for potential prenatal treatment.

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PubMed2005

Identification and characterization of engrafted human cells in human/goat xenogeneic transplantation chimerism.

We have injected human CD34+lin- cells derived from cord blood (CB) into the goat fetuses via in utero at 45-55 days gestation under guidance of B-scan ultrasonograph. Sixty out of 68 fetuses injected survived to full term. The long-term survival of the human cells in transplant goat has been tested by various experimental methods, including FACS analysis, real-time PCR, RT-PCR, Southern-blot hybridization, FISH, as well as immunohistochemical assays. All the 60 transplant goats demonstrated engrafted human cells, including myeloid, B-lymphoid, and erythroid lineages. The yield of the human CD34+ cells varied, but was not linked with sex and age. High numbers of human cells could be detected for at least 16 months after birth. Immunohistochemical analyses revealed that the human cells were present not only in blood but also in other tissues, such as liver, of the transplant goats. In addition, a human-specific serum albumin and the hepatocyte nuclear factor (hHNF-3beta) mRNAs specific to human hepato-antigen could be readily detected in the livers of the transplant goats. Our results demonstrate that this in utero xenograft model should be useful for expansion of human HSC and possibly for the evaluating the effectiveness of prenatal treatment of human genetic diseases.

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PubMed2004

Transcript profiling during preimplantation mouse development.

Studies using low-resolution methods to assess gene expression during preimplantation mouse development indicate that changes in gene expression either precede or occur concomitantly with the major morphological transitions, that is, conversion of the oocyte to totipotent 2-cell blastomeres, compaction, and blastocyst formation. Using microarrays, we characterized global changes in gene expression and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular processes that accompany and likely underlie these transitions. The analysis confirmed previously described processes or events, but more important, EASE revealed new insights. Response to DNA damage and DNA repair genes are overrepresented in the oocyte compared to 1-cell through blastocyst stages and may reflect the oocyte's response to selective pressures to insure genomic integrity; fertilization results in changes in the transcript profile in the 1-cell embryo that are far greater than previously recognized; and genome activation during 2-cell stage may not be as global and promiscuous as previously proposed, but rather far more selective, with genes involved in transcription and RNA processing being preferentially expressed. These results validate this hypothesis-generating approach by identifying genes involved in critical biological processes that can be the subject of a more traditional hypothesis-driven approach.

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PubMed2003

Gene expression in mouse oocytes and preimplantation embryos: use of suppression subtractive hybridization to identify oocyte- and embryo-specific genes.

The paucity of biological material has inhibited identifying genes that are differentially expressed during mammalian oogenesis and preimplantation development. We report here the linear amplification of mRNA from small numbers of mouse oocytes and preimplantation embryos to generate amounts of sense RNA that are sufficient for suppression subtractive hybridization. The resulting oocyte-specific and 8-cell-specific cDNA libraries were partially characterized, and the known oocyte-specific ZP1, ZP2, GDF-9, BMP15, and H1(oo) genes were found in the oocyte-specific cDNA library but not in the 8-cell-specific library. Further characterization of the subtracted oocyte and 8-cell embryo cDNA libraries should furnish a trove of information regarding temporal changes in gene expression during oogenesis and preimplantation development in the mouse.

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PubMed2002

[A study of the engraftment, expansion and differentiation of human hematopoietic stem cells in goats].

OBJECTIVE: To establish a human/goat hematopoietic stem cell (HSC) xenogeneic transplant model and to probe the engraftment, expansion and differentiation of human HSC in vivo. METHODS: Human HSCs were isolated from human umbilical cord blood and 1 x 10(5) human HSCs were in utero transplanted into 50 fetal goats at the 55 - 65 the gestation days. The engraftment, expansion and differentiation of human HSCs in goats were determined by FACS analysis, PCR and PCR-Southern blot hybridization at various intervals after birth. RESULTS: Hematopoietic chimerism occurred certainly in 35 of 39 live-born recipients. On average, The proportion of human hematopoietic cells in goat blood was 1% approximately 3% and remained phenotypically stable for at least 10 months. The human hematopoietic cells circulated in goat blood expressed CD34, CD14, CD20 and glycophorin A (GPA) but did not express CD3, CD4, CD7, CD8 and CD56 or expressed them at a very low level. CONCLUSION: The number of human HSC can be effectively expanded 1 000 - 10 000 fold. Human HSCs in goats undergo a limited differentiation. Our human/goat HSC xenogeneic model provides a useful way for the investigation of HSC transplantation, expansion and differentiation in vivo.

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PubMed2002

[The expression of human specific proteins in liver tissue of chimeric goats engrafted with human hematopoitic stem cells].

OBJECTIVE: To investigate the expression of human specific proteins in liver tissue obtained from goats engrafted with human hematopoietic stem cells (hHSC). METHODS: hHSCs derived from cord blood were transplanted into fetal goats for production of chimerism. Liver specimens were obtained from four 10-month-old chimeric goats and examined for their reactivity with monoclonal antibodies against human antigens: proliferating cell nuclear antigen (PCNA) and hepatocyte specific antigen (HSA). The presence of human HSA positive cells in the goat liver tissue was determined by fluorescence assisted cell sorting (FACS). The expression of human hepatocyte nuclear factor (hHNF-3beta) and human serum albumin (hALB) mRNAs was determined by RT-PCR. Meanwhile, FISH experiment was also performed to detect the human cells in the chimeric livers with the probe of human p17H8. RESULTS: Successful engraftment was confirmed by the detection of human blood cell chimerism in the circulation of the transplanted goats. Human PCNA and HSA were found in liver specimens of transplanted goats but not of normals. FACS analysis showed the presence of human HSA positive cells in the liver of chimeric goats. RT-PCR results demonstrated that hHNF-3beta and hALB mRNAs were specifically expressed in liver tissues of chimeric goats. FISH experiment showed two positive hybridized signals in some liver cells of the chimeric goats, indicating that human liver-like cells were present there. CONCLUSION: Goats engrafted with hHSC are capable to produce chimeric livers. Growth and propagation of human cells in the liver tissue of such transplanted goats were possible. Particularly, the human liver-like cells in chimeric goat livers had transcriptional activity specific to human hepatocytes.

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