Search PubMed⌕ Search

Biomedical subjects

Chang-Zheng Chen

Publications and source records attributed to Chang-Zheng Chen.

14 recordsLinked to original sources
PubMed2006

Methods for analyzing microRNA expression and function during hematopoietic lineage differentiation.

MicroRNAs (miRNAs), an abundant class of approx 22-nucleotide (nt) small RNAs that control gene expression at the posttranscriptional level, may play important roles during normal hematopoiesis and leukemogenesis. This chapter focuses on the methods and strategies for dissecting miRNA function during hematopoietic lineage differentiation. We describe a modified miRNA cloning method and expression analysis approach for determining miRNA expression during hematopoietic lineage differentiation. We illustrate a retroviral vector and a general strategy for the ectopic expression of miRNAs in hemato-poietic stem/progenitor cells. We discuss in vitro and in vivo functional assays that can be used to examine the roles of miRNAs during hematopoietic lineage differentiation. The methods and principles described here should also be applicable to study the roles of miRNAs in the differentiation and function of nonhematopoietic cell types.

Animals↗
PubMed2005

MicroRNAs as regulators of mammalian hematopoiesis.

MicroRNAs (miRNAs) are an abundant class of approximately 22 nucleotide non-coding RNAs and play important regulatory roles in animal and plant development at the post-transcriptional level. Many miRNAs cloned from mouse bone marrow cells are differentially regulated in various hematopoietic lineages, suggesting that they might influence hematopoietic lineage differentiation. miR-181, a miRNA specifically expressed in B cells within mouse bone marrow, promotes B-cell differentiation when expressed in hematopoietic stem/progenitor cells. Some human miRNAs are linked to leukemias: the miR-15a/miR-16 locus is frequently deleted or down-regulated in patients with B-cell chronic lymphocytic leukemia and miR-142 is at a translocation site found in a case of aggressive B-cell leukemia. Collectively, these results indicate that miRNAs may be important regulators of mammalian hematopoiesis. Here, we provide background on the biogenesis and function of miRNAs and discuss how miRNA-mediated post-transcriptional regulation may influence the development and function of blood cells.

Animals↗
PubMed2003

MicroRNAs modulate hematopoietic lineage differentiation.

MicroRNAs (miRNAs) are an abundant class of approximately 22-nucleotide regulatory RNAs found in plants and animals. Some miRNAs of plants, Caenorhabditis elegans, and Drosophila play important gene-regulatory roles during development by pairing to target mRNAs to specify posttranscriptional repression of these messages. We identify three miRNAs that are specifically expressed in hematopoietic cells and show that their expression is dynamically regulated during early hematopoiesis and lineage commitment. One of these miRNAs, miR-181, was preferentially expressed in the B-lymphoid cells of mouse bone marrow, and its ectopic expression in hematopoietic stem/progenitor cells led to an increased fraction of B-lineage cells in both tissue-culture differentiation assays and adult mice. Our results indicate that microRNAs are components of the molecular circuitry that controls mouse hematopoiesis and suggest that other microRNAs have similar regulatory roles during other facets of vertebrate development.

Animals↗
PubMed2003

The endoglin(positive) sca-1(positive) rhodamine(low) phenotype defines a near-homogeneous population of long-term repopulating hematopoietic stem cells.

Endoglin, an ancillary TGF-beta receptor, is differentially expressed in long-term repopulating hematopoietic stem cells (LTR-HSC). Here, we describe simple and highly efficient purification schemes for mouse bone marrow LTR-HSCs using Endoglin as a marker. The Endoglin positive and Sca-1 positive (Endo(Pos) Sca-1(Pos)) population, which contains about 36% of "Side Population" (SP) cells, is highly enriched for LTR-HSCs. In long-term competitive reconstitution assays, 100 such cells reconstituted all lethally irradiated recipients. Interestingly, the Endo(Pos) Sca-1(Pos) population contains comparable LTR-HSC activity in both SP and non-SP fractions, indicating that many HSCs are not captured by the SP phenotype. Furthermore, LTR-HSCs are exclusively found in the Endo(Pos) Sca-1(Pos) Lin(Neg/Low) (lineage negative/low), but not in the Endo(Neg) Sca-1(Pos) Lin(Neg/Low) population, suggesting that the Endo(Pos) population may contain all LTR-HSCs in mouse bone marrow. Finally, we demonstrated that the Endo(Pos) Sca-1(Pos) Rh(Low) (Rhodamine-123 low) phenotype, without using CD34, c-Kit, or Lineage markers, defines a nearly homogenous population of LTR-HSCs.

Animals↗
PubMed2002

Identification of endoglin as a functional marker that defines long-term repopulating hematopoietic stem cells.

We describe a strategy to obtain highly enriched long-term repopulating (LTR) hematopoietic stem cells (HSCs) from bone marrow side-population (SP) cells by using a transgenic reporter gene driven by a stem cell enhancer. To analyze the gene-expression profile of the rare HSC population, we developed an amplification protocol termed "constant-ratio PCR," in which sample and control cDNAs are amplified in the same PCR. This protocol allowed us to identify genes differentially expressed in the enriched LTR-HSC population by oligonucleotide microarray analysis using as little as 1 ng of total RNA. Endoglin, an ancillary transforming growth factor beta receptor, was differentially expressed by the enriched HSCs. Importantly, endoglin-positive cells, which account for 20% of total SP cells, contain all the LTR-HSC activity within bone marrow SP. Our results demonstrate that endoglin, which plays important roles in angiogenesis and hematopoiesis, is a functional marker that defines LTR HSCs. Our overall strategy may be applicable for the identification of markers for other tissue-specific stem cells.

Animals↗
PubMed1998

C-terminal His-tag Fusion Expression and Purification of Truncated cAMP-dependent Protein Kinase.

The truncated mCalpha lacking 3'-coding sequence of 96 base paires was fused with a His-tag (mCalpha4H) and a C-terminal fusion expression plasmid pZP mCalpha4H was constructed. With the induction of IPTG, the Expressed mCalpha4H was up to about 20% of the total bacterial proteins in E. coli BL21 (DE3). Using immobilized metal (Ni(2+)) chelation affinity chromatography, the target protein mCalpha4H was purified from crude lysates and inclusion bodies respectively. The results of in vitro and in vivo myristoylation assay showed that the purifed mCalpha4H is a substrate of NMT as the mCalpha.

Journal Article↗
PubMed1998

Selection of Myristoyltransferase Inhibitor Phages from Phage Display Random Peptide Library.

Through screening library with either immobilized or coated enzymes, we selected disease-related myristoyltransferase inhibitor phages from phage display random peptide library. After high-affinity bound phages were obtained, they were subjected to in vitro NMT inhibition assay to identify inhibitor phages. The results of DNA sequencing, peptide sequence deducing and sequence aligning of the 16 inhibitor phages suggested that the sequences of inhibitor peptides obtained by the two separate screening methods appeared overlapping, and included a consensus motif PX(0-3)H/R or H/RX(0-3)R, in which X represented a non-specific amino acid.

Journal Article↗
PubMed1997

His(6) Fusion Expression of Myristoyl-CoA: Protein N-myristoyltransferase in E. coli and its Purification.

Saccharomyces cerevisiae Myristoyl-CoA: protein N-myristoyltransferase (NMT) gene was cloned into a His(6)-fusion expression vector pMFHT. After transforming into E. coli BL21 (DE3), His(6)-NMT was induced to express at 37 degrees by IPTG. SDS-PAGE analysis showed an induced expression product band of about 54 kD which constituted about 10% of the total bacterial proteins. The analysis of product solubility revealed that His(6)-NMT was predominantly soluble. On the basis of these results, His(6)-NMT was purified in one-step to 95% of purity from bacterial lysates using immobilized metal (Ni(2+)) chelation affinity chromatography. The in vitro labelling experiment demonstrated that His(6)-NMT had an activity similar to that of mature NMT, suggesting that the His(6)-tag did not affect the enzyme activity. His(6)-tag in the N-terminal of NMT makes it be possible to immobilized simply on Ni(2+)-IDA Sepharose 6B resin, which can be used to screen the peptide inhibitors of NMT from Phage Display random peptide library.

Journal Article↗
PubMed1997

The Construction and Functional Study of Protein Kinase Inhibitor Phage.

A DNA fragment, which encoded the heat-stable protein kinase inhibitor (PKI) (5-24) of camp-dependent protein kinase (cAPK), was synthesized and cloned into phage display vector fe-tet-DOG1. Thus, PKI(5-24) was displayed on the surface of phage fd, which was termed PKI phage (cAPK inhibitor phage), in a form fused with gene III protein (g3p). It was showed that PKI phage not only repressed cAPK effectively, but also bound with the immobilized recombinant His(6)-tag mouse cAPK-Calpha( His(6)-mCalpha) specifically. The bound PKI phages could be quantitatively eluted under acidic conditions. Model affinity screening demonstrated that PKI phages could be selectively enriched from the mixture of PKI phage and wild-type phages (1:10(8)) using affinity chromatography of immobilized His(6)-mCalpha. These results suggest that selecting protein kinase inhibitor by phage display technique is feasible.

Journal Article↗
PubMed1996

The Construction of T7 Promoter-based His(6)-tagging Vectors and the Single-step Purification of the Expression products.

T7 promoter-based fusion expression vectors have been constructed that directed the synthesis of heterologous proteins in E. coli as fusions with a stretch of six consecutive histidine residues His(6) at N Terminus. The vectors were also featured with strong T7 promoter, terminator, translational start, multiple cloning sites with seven unique restriction sites in all three reading frames and the f1 phage origin which allows the packaging of single-stranded plasmid, mutagenesis and DNA sequencing without subcloning steps. In most cases, expressed fusion proteins are soluble. The His(6) tag allows the fusion proteins purified in one step by immobilized metal (Ni(2+)) chelation affinity chromatography in the denatured or native state. As an example of the general utility of these expression vectors, the His(6)-fused catalytic subunits of mouse cAMP-dependent protein kinase were expressed with high activity by using these vectors and could be purified to homogeneity in one step.

Journal Article↗
PubMed1996

A T7 Promoter-based Versatile Expression Vector System.

A family of T7 promoter-based versatile expression plamides for E. coli Were constructed. These vectors were featured strong T7 promoter, translational start, stop elements, a multiple cloning site with eight unique restriction sites in all three reading frames and f1 phage origin which allows packaging of single stranded plasmid, mutagensis and gene sequencing without subcloning steps. With these vectors recombinant proteins can be expressed in mature or short fusion forms. Many heterolegous genes have been highly expressed using these vectors, most of them were expressed in soluble and active forms.

Journal Article↗
PubMed1996

Cloning and Expression of the Yeast Saccharomyces cerevisiae MyristoyL-CoA: Protein N-Myristoyltransferase Gene.

The gene encoding the myristoyl-CoA: protein N-myristoyltransferase from yeast S. cerevisiae (YSCNMT) has been amplified with the synthetic oligonucleotides by PCR, cloned into the pBluescript KS+ vector and analyzed by DNA sequencing. Then, an expression plasmid (pMFT7-5-NMT) containing the cloned YSCNMT gene controlled by T7 promoter has been constructed and transformed into E. coli BL21(DE3). Analyzed by SDS-PAGE, an IPTG-induced product with the same molecular size (53 kD) as that deduced from the known YSCNMT amino acid sequence was observed. It accounted for about 39% of the total cellular proteins, the soluble form of which accounted for about 34% of the total soluble cellular proteins and was purified to homogeneity in a single step using P11 phosphocellulose chromatography. The N-terminal amino acid sequencing revealed that the expressed protein began with the sequence encoded by the cloned YSCNMT gene and did not contain the N-terminal methionine. It was also observed that the expressed product had apparent YSCNMT activity in the in vitro assay.

Journal Article↗