ee-chun cheng - Academia.edu (original) (raw)
Papers by ee-chun cheng
Additional file 3: Fig. S3. Maternal and zygotic PUM1 are required for postnatal survival. The to... more Additional file 3: Fig. S3. Maternal and zygotic PUM1 are required for postnatal survival. The top panel shows the crosses observed over at least a 6 month period. n = number of matings pairs. %P0/P1 lethality is the number of pups that are born dead at birth or after 1 day after birth. Pups/litter–mean (SD).
Additional file 2: Fig. S2. Two-cell m−z− and m−z+ embryos have more similar transcriptomes to ea... more Additional file 2: Fig. S2. Two-cell m−z− and m−z+ embryos have more similar transcriptomes to each other than to m+z+ embryos. RNA seq analysis was performed on m+z+ (WT) two-cell embryos from Pum1+/+ reciprocal matings, m−z− (KO) two-cell embryos from Pum1−/− reciprocal matings and m−z+ (HET) two-cell embryos from Pum1−/− females mated with Pum1+/+ male. A The heatmap of Spearman correlation coefficient between the different oocytes and two-cell embryos. B–D Shows the scatterplot for the comparisons of the different two-cell transcriptomes.
Additional file 1: Fig. S1. Aâ D Shows the comparison of RNA-seq data with previous microarray st... more Additional file 1: Fig. S1. Aâ D Shows the comparison of RNA-seq data with previous microarray study of mRNA transcripts changed from GV to MII (Su et al. [3]). *pâ
Cell Reports, 2021
SETDB1 is a histone-lysine N-methyltransferase critical for germline development. However, its fu... more SETDB1 is a histone-lysine N-methyltransferase critical for germline development. However, its function in early meiotic prophase I remains unknown. Here, we report that Setdb1 null spermatocytes display aberrant centromere clustering during leptotene, bouquet formation during zygotene, and subsequent failure in pairing and synapsis of homologous chromosomes, as well as compromised meiotic silencing of unsynapsed chromatin, which leads to meiotic arrest before pachytene and apoptosis of spermatocytes. H3K9me3 is enriched in centromeric or pericentromeric regions and is present in many sites throughout the genome, with a subset changed in the Setdb1 mutant. These observations indicate that SETDB1-mediated H3K9me3 is essential for the bivalent formation in early meiosis. Transcriptome analysis reveals the function of SETDB1 in repressing transposons and transposon-proximal genes and in regulating meiotic and somatic lineage genes. These findings highlight a mechanism in which SETDB1-mediated H3K9me3 during early meiosis ensures the formation of homologous bivalents and survival of spermatocytes.
Cell & Bioscience, 2018
Genome research, 2015
The eukaryotic genome has vast intergenic regions containing transposons, pseudogenes, and other ... more The eukaryotic genome has vast intergenic regions containing transposons, pseudogenes, and other repetitive sequences. They produce numerous long noncoding RNAs (lncRNAs) and Piwi-interacting RNAs (piRNAs), yet the functions of the vast intergenic regions remain largely unknown. Mammalian piRNAs are abundantly expressed from the spermatocyte to round spermatid stage, coinciding with the widespread expression of lncRNAs in these cells. Here, we show that piRNAs derived from transposons and pseudogenes mediate the degradation of a large number of mRNAs and lncRNAs in mouse late spermatocytes. In particular, they have a large impact on the lncRNA transcriptome, as a quarter of lncRNAs expressed in late spermatocytes are up-regulated in mice deficient in the piRNA pathway. Furthermore, our genomic and in vivo functional analyses reveal that retrotransposon sequences in the 3' UTR of mRNAs are targeted by piRNAs for degradation. Similarly, the degradation of spermatogenic cell-specif...
PloS one, 2014
PIWI proteins play essential and conserved roles in germline development, including germline stem... more PIWI proteins play essential and conserved roles in germline development, including germline stem cell maintenance and meiosis. Because germline regulators such as OCT4, NANOG, and SOX2 are known to be potent factors that reprogram differentiated somatic cells into induced pluripotent stem cells (iPSCs), we investigated whether the PIWI protein family is involved in iPSC production. We find that all three mouse Piwi genes, Miwi, Mili, and Miwi2, are expressed in embryonic stem cells (ESCs) at higher levels than in fibroblasts, with Mili being the highest. However, mice lacking all three Piwi genes are viable and female fertile, and are only male sterile. Furthermore, embryonic fibroblasts derived from Miwi/Mili/Miwi2 triple knockout embryos can be efficiently reprogrammed into iPS cells. These iPS cells expressed pluripotency markers and were capable of differentiating into all three germ layers in teratoma assays. Genome-wide expression profiling reveals that the triple knockout iP...
RNA, 2010
Loss-of-function studies in human embryonic stem cells (hESCs) and induced pluripotent stem cells... more Loss-of-function studies in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) via nonviral approaches have been largely unsuccessful. Here we report a simple and cost-effective method for high-efficiency delivery of plasmids and siRNAs into hESCs and iPSCs. Using this method for siRNA delivery, we achieve >90% reduction in the expression of the stem cell factors Oct4 and Lin28, and observe cell morphological and staining pattern changes, characteristics of hESC differentiation, as a result of Oct4 knockdown.
Blood, 2009
Megakaryoblastic leukemia 1 (MKL1), identified as part of the t(1;22) translocation specific to a... more Megakaryoblastic leukemia 1 (MKL1), identified as part of the t(1;22) translocation specific to acute megakaryoblastic leukemia, is highly expressed in differentiated muscle cells and promotes muscle differentiation by activating serum response factor (SRF). Here we show that Mkl1 expression is up-regulated during murine megakaryocytic differentiation and that enforced overexpression of MKL1 enhances megakaryocytic differentiation. When the human erythroleukemia (HEL) cell line is induced to differentiate with 12-O-tetradecanoylphorbol 13-acetate, overexpression of MKL1 results in an increased number of megakaryocytes with a concurrent increase in ploidy. MKL1 overexpression also promotes megakaryocytic differentiation of primary human CD34+ cells cultured in the presence of thrombopoietin. The effect of MKL1 is abrogated when SRF is knocked down, suggesting that MKL1 acts through SRF. Consistent with these findings in human cells, knockout of Mkl1 in mice leads to reduced platelet ...
SSRN Electronic Journal, 2022
Blood, 2008
The OTT-MKL1 fusion gene product is generated as a result of t(1;22) in a subset of acute megakar... more The OTT-MKL1 fusion gene product is generated as a result of t(1;22) in a subset of acute megakaryoblastic leukemia predominantly encountered in young children. Due to myelofibrosis and the age at presentation, patient samples are scarce. We generated Human Erythroid Leukemia (HEL) cell derived cell lines with tet-inducible OTT, MKL1 and OTT-MKL1 to further elucidate the function of the respective proteins. HEL cells can be induced to differentiate down the megakaryocyte lineage by TPA. Induction with doxycycline resulted in transcription and translation of the respective genes within hours. While overexpression of MKL1 led to enhancement of megakaryocytic differentiation, both OTT and OTT-MKL1 overexpression led to cell death over the course of several days by apoptosis as evident by staining for Annexin V and morphology. The apoptotic cell death was greatly enhanced by concomitant induction of differentiation by TPA. We performed microarray analysis comparing uninduced and 8-hour ...
Blood, 2007
Acute Megakaryoblastic Leukemia (AMKL or AML variant M7), which occurs most often in infants and ... more Acute Megakaryoblastic Leukemia (AMKL or AML variant M7), which occurs most often in infants and young children, is characterized by a failure of megakaryocyte (MK) differentiation, bone marrow fibrosis, cytogenetic abnormalities, and a poor prognosis. We are particularly interested in AMKL that is associated with the translocation t(1;22)(p13;q13), which yields an in-frame fusion of RBM15 (OTT) and MKL1 (MAL) on chromosomes 1 and 22, respectively. The resultant fusion, RBM15-MKL1 is believed to include all of the functional domains of each component. In order to better understand the role of RBM15-MKL1 in AMKL, it is necessary to understand the roles of the constituent genes, RBM15 and MKL1, in hematopoiesis. We have studied the role of human MKL1 in megakaryopoiesis using primary human CD34+ cells purified from G-CSF mobilized PBMC from healthy donors (n=4). To optimize the CD34+ model, we tested the ability of TPO vs. TPO+SCF vs. TPO+SCF+IL–3 to induce megakaryocytopoiesis. TPO a...
Blood, 2007
Our studies demonstrate a critical role for MKL1 (megakaryoblastic leukemia 1) in the molecular r... more Our studies demonstrate a critical role for MKL1 (megakaryoblastic leukemia 1) in the molecular regulation of megakaryocytopoiesis. MKL1 is part of the fusion protein formed by the t (1; 22) translocation, which is found uniquely in Acute Megakaryoblastic Leukemia (AMKL). The translocation fuses the RBM15 (also known as OTT) gene on chromosome 1 with the MKL1 (also known as MAL) gene on chromosome 22. Previous studies in muscle cells show that MKL1 is a positive cofactor for the transcription factor serum response factor (SRF), and works via the Rho-A pathway to turn on immediate early genes and muscle specific genes. Using qRT-PCR we show that MKL1 mRNA is markedly up-regulated during megakaryocyte (MK) differentiation of primary murine bone marrow and fetal liver cells. When we overexpress MKL1 in the human erythroleukemia (HEL) cell line and differentiate the cells to become MK by phorbol ester (TPA), there is far greater MK differentiation than in control HEL cells. Via analysis...
PloS one, 2015
Malformin C, a fungal cyclic pentapeptide, has been claimed to have anti-cancer potential, but no... more Malformin C, a fungal cyclic pentapeptide, has been claimed to have anti-cancer potential, but no in vivo study was available to substantiate this property. Therefore, we conducted in vitro and in vivo experiments to investigate its anti-cancer effects and toxicity. Our studies showed Malformin C inhibited Colon 38 and HCT 116 cell growth dose-dependently with an IC50 of 0.27±0.07μM and 0.18±0.023μM respectively. This inhibition was explicated by Malformin C's effect on G2/M arrest. Moreover, we observed up-regulated expression of phospho-histone H2A.X, p53, cleaved CASPASE 3 and LC3 after Malformin C treatment, while the apoptosis assay indicated an increased population of necrotic and late apoptotic cells. In vivo, the pathological study exhibited the acute toxicity of Malformin C at lethal dosage in BDF1 mice might be caused by an acute yet subtle inflammatory response, consistent with elevated IL-6 in the plasma cytokine assay. Further anti-tumor and toxicity experiments pro...
PloS one, 2013
Hematopoietic stem cells (HSC) must engage in a life-long balance between self-renewal and differ... more Hematopoietic stem cells (HSC) must engage in a life-long balance between self-renewal and differentiation to sustain hematopoiesis. The highly conserved PIWI protein family regulates proliferative states of stem cells and their progeny in diverse organisms. A Human piwi gene (for clarity, the non-italicized "piwi" refers to the gene subfamily), HIWI (PIWIL1), is expressed in CD34⁺ stem/progenitor cells and transient expression of HIWI in a human leukemia cell line drastically reduces cell proliferation, implying the potential function of these proteins in hematopoiesis. Here, we report that one of the three piwi genes in mice, Miwi2 (Piwil4), is expressed in primitive hematopoetic cell types within the bone marrow. Mice with a global deletion of all three piwi genes, Miwi, Mili, and Miwi2, are able to maintain long-term hematopoiesis with no observable effect on the homeostatic HSC compartment in adult mice. The PIWI-deficient hematopoetic cells are capable of normal line...
Transgenic Research, 2007
Green fluorescent protein (GFP) transgenic animals are widely used in biomedical research. We obs... more Green fluorescent protein (GFP) transgenic animals are widely used in biomedical research. We observed that the commonly used beta-actin-GFP transgenic mouse has renal defects with proteinuria starting as early as 5 weeks of age. Histological analysis reveals a widespread increase in glomerular extracellular matrix, occasional mesangiolysis, and secondary tubulointerstitial injury. Electron microscopic (EM) analysis reveals dramatic thickening of the glomerular basement membrane (GBM). Several other transgenic strains with GFP on ubiquitous promoters including beta-actin (with insertion in a different location) and ubiquitin C show no renal abnormalities. Western blot analysis on crude glomerular preparations from several GFP transgenic strains revealed that higher levels of GFP expression might be responsible for the observed pathogenesis. Mapping of the transgene insertion site by inverse PCR indicates that the beta-actin GFP transgene does not cause insertional mutagenesis nor does it modify the transcription level of adjacent genes. Taken together, this strain of beta-actin-GFP transgenic mouse may be used to study the mechanism of GBM expansion. Moreover, experiments using this strain of GFP mouse should be hereafter carefully planned because its renal pathology may interfere with data interpretation.
Tissue Engineering Part A, 2009
Stem Cells, 2013
Discovery of the cellular and molecular mechanisms of induced pluripotency has been hampered by i... more Discovery of the cellular and molecular mechanisms of induced pluripotency has been hampered by its low efficiency and slow kinetics. Here, we report an experimental system with multicolor time-lapse microscopy that permits direct observation of pluripotency induction at single cell resolution, with temporal intervals as short as 5 minutes. Using granulocyte-monocyte progenitors as source cells, we visualized nascent pluripotent cells that emerge from a hematopoietic state. We engineered a suite of image processing and analysis software to annotate the behaviors of the reprogramming cells, which revealed the highly dynamic cell-cell interactions associated with early reprogramming. We observed frequent cell migration, which can lead to sister colonies, satellite colonies, and colonies of mixed genetic makeup. In addition, we discovered a previously unknown morphologically distinct two-cell intermediate of reprogramming, which occurs prior to other reprogramming landmarks. By directl...
Additional file 3: Fig. S3. Maternal and zygotic PUM1 are required for postnatal survival. The to... more Additional file 3: Fig. S3. Maternal and zygotic PUM1 are required for postnatal survival. The top panel shows the crosses observed over at least a 6 month period. n = number of matings pairs. %P0/P1 lethality is the number of pups that are born dead at birth or after 1 day after birth. Pups/litter–mean (SD).
Additional file 2: Fig. S2. Two-cell m−z− and m−z+ embryos have more similar transcriptomes to ea... more Additional file 2: Fig. S2. Two-cell m−z− and m−z+ embryos have more similar transcriptomes to each other than to m+z+ embryos. RNA seq analysis was performed on m+z+ (WT) two-cell embryos from Pum1+/+ reciprocal matings, m−z− (KO) two-cell embryos from Pum1−/− reciprocal matings and m−z+ (HET) two-cell embryos from Pum1−/− females mated with Pum1+/+ male. A The heatmap of Spearman correlation coefficient between the different oocytes and two-cell embryos. B–D Shows the scatterplot for the comparisons of the different two-cell transcriptomes.
Additional file 1: Fig. S1. Aâ D Shows the comparison of RNA-seq data with previous microarray st... more Additional file 1: Fig. S1. Aâ D Shows the comparison of RNA-seq data with previous microarray study of mRNA transcripts changed from GV to MII (Su et al. [3]). *pâ
Cell Reports, 2021
SETDB1 is a histone-lysine N-methyltransferase critical for germline development. However, its fu... more SETDB1 is a histone-lysine N-methyltransferase critical for germline development. However, its function in early meiotic prophase I remains unknown. Here, we report that Setdb1 null spermatocytes display aberrant centromere clustering during leptotene, bouquet formation during zygotene, and subsequent failure in pairing and synapsis of homologous chromosomes, as well as compromised meiotic silencing of unsynapsed chromatin, which leads to meiotic arrest before pachytene and apoptosis of spermatocytes. H3K9me3 is enriched in centromeric or pericentromeric regions and is present in many sites throughout the genome, with a subset changed in the Setdb1 mutant. These observations indicate that SETDB1-mediated H3K9me3 is essential for the bivalent formation in early meiosis. Transcriptome analysis reveals the function of SETDB1 in repressing transposons and transposon-proximal genes and in regulating meiotic and somatic lineage genes. These findings highlight a mechanism in which SETDB1-mediated H3K9me3 during early meiosis ensures the formation of homologous bivalents and survival of spermatocytes.
Cell & Bioscience, 2018
Genome research, 2015
The eukaryotic genome has vast intergenic regions containing transposons, pseudogenes, and other ... more The eukaryotic genome has vast intergenic regions containing transposons, pseudogenes, and other repetitive sequences. They produce numerous long noncoding RNAs (lncRNAs) and Piwi-interacting RNAs (piRNAs), yet the functions of the vast intergenic regions remain largely unknown. Mammalian piRNAs are abundantly expressed from the spermatocyte to round spermatid stage, coinciding with the widespread expression of lncRNAs in these cells. Here, we show that piRNAs derived from transposons and pseudogenes mediate the degradation of a large number of mRNAs and lncRNAs in mouse late spermatocytes. In particular, they have a large impact on the lncRNA transcriptome, as a quarter of lncRNAs expressed in late spermatocytes are up-regulated in mice deficient in the piRNA pathway. Furthermore, our genomic and in vivo functional analyses reveal that retrotransposon sequences in the 3' UTR of mRNAs are targeted by piRNAs for degradation. Similarly, the degradation of spermatogenic cell-specif...
PloS one, 2014
PIWI proteins play essential and conserved roles in germline development, including germline stem... more PIWI proteins play essential and conserved roles in germline development, including germline stem cell maintenance and meiosis. Because germline regulators such as OCT4, NANOG, and SOX2 are known to be potent factors that reprogram differentiated somatic cells into induced pluripotent stem cells (iPSCs), we investigated whether the PIWI protein family is involved in iPSC production. We find that all three mouse Piwi genes, Miwi, Mili, and Miwi2, are expressed in embryonic stem cells (ESCs) at higher levels than in fibroblasts, with Mili being the highest. However, mice lacking all three Piwi genes are viable and female fertile, and are only male sterile. Furthermore, embryonic fibroblasts derived from Miwi/Mili/Miwi2 triple knockout embryos can be efficiently reprogrammed into iPS cells. These iPS cells expressed pluripotency markers and were capable of differentiating into all three germ layers in teratoma assays. Genome-wide expression profiling reveals that the triple knockout iP...
RNA, 2010
Loss-of-function studies in human embryonic stem cells (hESCs) and induced pluripotent stem cells... more Loss-of-function studies in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) via nonviral approaches have been largely unsuccessful. Here we report a simple and cost-effective method for high-efficiency delivery of plasmids and siRNAs into hESCs and iPSCs. Using this method for siRNA delivery, we achieve >90% reduction in the expression of the stem cell factors Oct4 and Lin28, and observe cell morphological and staining pattern changes, characteristics of hESC differentiation, as a result of Oct4 knockdown.
Blood, 2009
Megakaryoblastic leukemia 1 (MKL1), identified as part of the t(1;22) translocation specific to a... more Megakaryoblastic leukemia 1 (MKL1), identified as part of the t(1;22) translocation specific to acute megakaryoblastic leukemia, is highly expressed in differentiated muscle cells and promotes muscle differentiation by activating serum response factor (SRF). Here we show that Mkl1 expression is up-regulated during murine megakaryocytic differentiation and that enforced overexpression of MKL1 enhances megakaryocytic differentiation. When the human erythroleukemia (HEL) cell line is induced to differentiate with 12-O-tetradecanoylphorbol 13-acetate, overexpression of MKL1 results in an increased number of megakaryocytes with a concurrent increase in ploidy. MKL1 overexpression also promotes megakaryocytic differentiation of primary human CD34+ cells cultured in the presence of thrombopoietin. The effect of MKL1 is abrogated when SRF is knocked down, suggesting that MKL1 acts through SRF. Consistent with these findings in human cells, knockout of Mkl1 in mice leads to reduced platelet ...
SSRN Electronic Journal, 2022
Blood, 2008
The OTT-MKL1 fusion gene product is generated as a result of t(1;22) in a subset of acute megakar... more The OTT-MKL1 fusion gene product is generated as a result of t(1;22) in a subset of acute megakaryoblastic leukemia predominantly encountered in young children. Due to myelofibrosis and the age at presentation, patient samples are scarce. We generated Human Erythroid Leukemia (HEL) cell derived cell lines with tet-inducible OTT, MKL1 and OTT-MKL1 to further elucidate the function of the respective proteins. HEL cells can be induced to differentiate down the megakaryocyte lineage by TPA. Induction with doxycycline resulted in transcription and translation of the respective genes within hours. While overexpression of MKL1 led to enhancement of megakaryocytic differentiation, both OTT and OTT-MKL1 overexpression led to cell death over the course of several days by apoptosis as evident by staining for Annexin V and morphology. The apoptotic cell death was greatly enhanced by concomitant induction of differentiation by TPA. We performed microarray analysis comparing uninduced and 8-hour ...
Blood, 2007
Acute Megakaryoblastic Leukemia (AMKL or AML variant M7), which occurs most often in infants and ... more Acute Megakaryoblastic Leukemia (AMKL or AML variant M7), which occurs most often in infants and young children, is characterized by a failure of megakaryocyte (MK) differentiation, bone marrow fibrosis, cytogenetic abnormalities, and a poor prognosis. We are particularly interested in AMKL that is associated with the translocation t(1;22)(p13;q13), which yields an in-frame fusion of RBM15 (OTT) and MKL1 (MAL) on chromosomes 1 and 22, respectively. The resultant fusion, RBM15-MKL1 is believed to include all of the functional domains of each component. In order to better understand the role of RBM15-MKL1 in AMKL, it is necessary to understand the roles of the constituent genes, RBM15 and MKL1, in hematopoiesis. We have studied the role of human MKL1 in megakaryopoiesis using primary human CD34+ cells purified from G-CSF mobilized PBMC from healthy donors (n=4). To optimize the CD34+ model, we tested the ability of TPO vs. TPO+SCF vs. TPO+SCF+IL–3 to induce megakaryocytopoiesis. TPO a...
Blood, 2007
Our studies demonstrate a critical role for MKL1 (megakaryoblastic leukemia 1) in the molecular r... more Our studies demonstrate a critical role for MKL1 (megakaryoblastic leukemia 1) in the molecular regulation of megakaryocytopoiesis. MKL1 is part of the fusion protein formed by the t (1; 22) translocation, which is found uniquely in Acute Megakaryoblastic Leukemia (AMKL). The translocation fuses the RBM15 (also known as OTT) gene on chromosome 1 with the MKL1 (also known as MAL) gene on chromosome 22. Previous studies in muscle cells show that MKL1 is a positive cofactor for the transcription factor serum response factor (SRF), and works via the Rho-A pathway to turn on immediate early genes and muscle specific genes. Using qRT-PCR we show that MKL1 mRNA is markedly up-regulated during megakaryocyte (MK) differentiation of primary murine bone marrow and fetal liver cells. When we overexpress MKL1 in the human erythroleukemia (HEL) cell line and differentiate the cells to become MK by phorbol ester (TPA), there is far greater MK differentiation than in control HEL cells. Via analysis...
PloS one, 2015
Malformin C, a fungal cyclic pentapeptide, has been claimed to have anti-cancer potential, but no... more Malformin C, a fungal cyclic pentapeptide, has been claimed to have anti-cancer potential, but no in vivo study was available to substantiate this property. Therefore, we conducted in vitro and in vivo experiments to investigate its anti-cancer effects and toxicity. Our studies showed Malformin C inhibited Colon 38 and HCT 116 cell growth dose-dependently with an IC50 of 0.27±0.07μM and 0.18±0.023μM respectively. This inhibition was explicated by Malformin C's effect on G2/M arrest. Moreover, we observed up-regulated expression of phospho-histone H2A.X, p53, cleaved CASPASE 3 and LC3 after Malformin C treatment, while the apoptosis assay indicated an increased population of necrotic and late apoptotic cells. In vivo, the pathological study exhibited the acute toxicity of Malformin C at lethal dosage in BDF1 mice might be caused by an acute yet subtle inflammatory response, consistent with elevated IL-6 in the plasma cytokine assay. Further anti-tumor and toxicity experiments pro...
PloS one, 2013
Hematopoietic stem cells (HSC) must engage in a life-long balance between self-renewal and differ... more Hematopoietic stem cells (HSC) must engage in a life-long balance between self-renewal and differentiation to sustain hematopoiesis. The highly conserved PIWI protein family regulates proliferative states of stem cells and their progeny in diverse organisms. A Human piwi gene (for clarity, the non-italicized "piwi" refers to the gene subfamily), HIWI (PIWIL1), is expressed in CD34⁺ stem/progenitor cells and transient expression of HIWI in a human leukemia cell line drastically reduces cell proliferation, implying the potential function of these proteins in hematopoiesis. Here, we report that one of the three piwi genes in mice, Miwi2 (Piwil4), is expressed in primitive hematopoetic cell types within the bone marrow. Mice with a global deletion of all three piwi genes, Miwi, Mili, and Miwi2, are able to maintain long-term hematopoiesis with no observable effect on the homeostatic HSC compartment in adult mice. The PIWI-deficient hematopoetic cells are capable of normal line...
Transgenic Research, 2007
Green fluorescent protein (GFP) transgenic animals are widely used in biomedical research. We obs... more Green fluorescent protein (GFP) transgenic animals are widely used in biomedical research. We observed that the commonly used beta-actin-GFP transgenic mouse has renal defects with proteinuria starting as early as 5 weeks of age. Histological analysis reveals a widespread increase in glomerular extracellular matrix, occasional mesangiolysis, and secondary tubulointerstitial injury. Electron microscopic (EM) analysis reveals dramatic thickening of the glomerular basement membrane (GBM). Several other transgenic strains with GFP on ubiquitous promoters including beta-actin (with insertion in a different location) and ubiquitin C show no renal abnormalities. Western blot analysis on crude glomerular preparations from several GFP transgenic strains revealed that higher levels of GFP expression might be responsible for the observed pathogenesis. Mapping of the transgene insertion site by inverse PCR indicates that the beta-actin GFP transgene does not cause insertional mutagenesis nor does it modify the transcription level of adjacent genes. Taken together, this strain of beta-actin-GFP transgenic mouse may be used to study the mechanism of GBM expansion. Moreover, experiments using this strain of GFP mouse should be hereafter carefully planned because its renal pathology may interfere with data interpretation.
Tissue Engineering Part A, 2009
Stem Cells, 2013
Discovery of the cellular and molecular mechanisms of induced pluripotency has been hampered by i... more Discovery of the cellular and molecular mechanisms of induced pluripotency has been hampered by its low efficiency and slow kinetics. Here, we report an experimental system with multicolor time-lapse microscopy that permits direct observation of pluripotency induction at single cell resolution, with temporal intervals as short as 5 minutes. Using granulocyte-monocyte progenitors as source cells, we visualized nascent pluripotent cells that emerge from a hematopoietic state. We engineered a suite of image processing and analysis software to annotate the behaviors of the reprogramming cells, which revealed the highly dynamic cell-cell interactions associated with early reprogramming. We observed frequent cell migration, which can lead to sister colonies, satellite colonies, and colonies of mixed genetic makeup. In addition, we discovered a previously unknown morphologically distinct two-cell intermediate of reprogramming, which occurs prior to other reprogramming landmarks. By directl...