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  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

• the present invention relates to a RNA molecule, particularly to an endogenous short hairpin RNA as well as the application of the invented RNA to induce the formation of hematopoietic cells by repressing ElA-like inhibitor of differentiation- 1 (referred to hereinafter as EIDl).
• EIDl ElA-like inhibitor of differentiation- 1
• BMSCs bone marrow mesenchymal stem cells
• HSCs hematopoietic stem cells
• BMSCs expanded in culture can be distinguished from HSCs by the lack of expression of CD45 and CD34 (G. Chamberlain, J. Fox, B. Ashton, J. Middleton, Stem Cells. 25, 2739-49 (2007).). Since it is a great challenge to obtain a large number of HSCs for clinical application (B.P. Sorrentino, Nat Rev Immunol.
• BMSCs would serve as an alternate source for HSCs if BMSCs can be induced to differentiate into hematopoietic cells.
• mesenchymal stem cells BMSCs
• BMSCs mesenchymal stem cells
• HSCs hematopoietic stem cells
• miRNAs are differentially expressed in different hematopoietic cell types and play important regulatory roles.
• miR-181 is associated with B lymphoid development (CZ. Chen et al., Science 303, 83-86 (2004)), miRNA-142 and -223 with T lymphopoiesis (S. H. Ramkissoon et al., Leukemia Research, 30, 643-647 (2005).), miRNA-221 and -222 with erythropoiesis (N. Felli et al., Proc Natl Acad Sci U S A. 102, 18081-18086 (2005).), miRNA-223 with granulocytic differentiation (F.
• miRNAs have been shown to prevent the differentiation of HSCs (III, R. W. Georgantas et al., Proc Natl Acad Sci U S A.104, 2750-2755 (2007).).
• miRNAs such as miR-130a and miR-10a have been found to target the transcription factor genes HOXAl and MAFB, which are important for cellular differentiation (R.
• the first object of the invention is to provide an endogenous short hairpin RNA or a complement thereof having a sequence of SEQ. ID. NO.: 1 that induces the formation of hematopoietic cells particularly by repressing EIDl.
• the said SEQ. ID. NO.: 1 shows:
• the said sequence of SEQ. ID. NO.: 1 essentially includes the nucleic acid of 5'-CAA AUA CUC A-3'.
• the second object of the invention is to provide an expression construct including the sequence of SEQ. ID. NO.: 1.
• the third object of the invention is to provide a vector comprising an expression construct having an expression construct including the sequence of SEQ. ID. NO.: 1.
• the vector preferably is a retrovirus plasmid (retrovirus vector pMSCV ) that expresses the sequence of SEQ. ID. NO.: 1.
• the fourth object of the invention is to provide an application of the invented RNA or a complement thereof having a sequence of SEQ. ID. NO.: 1 to induce the formation of hematopoietic cells or a method of treatment for a subject suffering from dysfunction of formation of blood cells or a method of treatment for hematopoiesis in mammalian including human.
• the invention also provides a use of an endogenous short hairpin RNA having SEQ. ID. NO.: 1 or SEQ. ID. NO.: 2 in manufacture of medicament for treatment of a subject suffering from dysfunction of hematopoiesis in mammalian including human.
• the said SEQ. ID. NO.: 2 shows 5'-UGU AAC ACA AUG GUG AGU
• the query sequence is at most 23 nucleotides in length.
• the sequence is at least 10 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at most 23 nucleotides. And the GAP analysis aligns the two sequences over a region of at least 10 nucleotides.
• a polynucleotide of the invention comprises a sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99
• Polynucleotides of the present invention are natural ones, and may possess one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site- directed mutagenesis on the nucleic acid).
• Oligonucleotides and/or polynucleotides of the invention hybridize to a sill- gene of the present invention, or a region flanking said gene, under stringent conditions.
• stringent hybridization conditions refers to parameters with which the art is familiar, including the variation of the hybridization temperature with length of an oligonucleotide. Nucleic acid hybridization parameters may be found in references which compile such methods, Sambrook, et al. (supra), and Ausubel, et al. (supra).
• stringent hybridization conditions can refer to hybridization at 65°C in hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin (BSA), 2.5 mM NaH2PO4 (pH7), 0.5% SDS, 2 niM EDTA), followed by one or more washes in 0.2.xSSC, 0.01% BSA at 500C.
• hybridization buffer 3.5xSSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin (BSA), 2.5 mM NaH2PO4 (pH7), 0.5% SDS, 2 niM EDTA
• nucleic acid and/or oligonucleotides hybridize to the region of the an insect genome of interest, such as the genome of a honeybee, under conditions used in nucleic acid amplification techniques such as PCR.
• BMSCs transduced with a newly identified short hairpin RNA (shRNA) of the present invention can differentiate into hematopoietic stem cells (HSCs) and their descendant multipotent progenitor cells that have the capacity of further differentiating into blood cells in vitro, according to the invention, on transplantation into sublethally irradiated NOD/SCID mice, transduced human BMSCs engrafted and differentiated into all hematopoietic lineages including lymphocytes and myelocytes. Furthermore this new shRNA alters BMSC fate by repressing the translation of EIDl.
• shRNA short hairpin RNA
• shRNAs Like artificial shRNAs, the two strands of their stems are perfectly complementary with a length of at least 21 nucleotides.
• shRNAs important in regulating the self-renewal and directional differentiation of stem cells we employed a custom microarray for a high-throughput screen (see. table 1).
• sRNA BMSC
• HSC HSC/BMSC
• *ZK-249 is referred as shR-CH in this paper.
• shR-CH The expression of shR-CH was found to be at least 10 times higher in HSCs than in MSCs. To determine the length of mature shR-CH, this endogenous shRNA has been cloned. As shown in Figure 2B, mature shR-CH was about 21nt in length and its precursor formed a perfectly complementary hairpin structure. The said shR-CH resides within the first intron of SH3PXD2B gene that is located on chromosome 5 and is not phylogenetically conserved as most endogenous shRNAs (17 Accompanying manuscript).
• EIDl (ElA-like inhibitor of differentiation- I)(NM O 14335.2) was selected for validation by qRT-PCR and Western blot assay because it contains five naturally-occurring putative shR-CH binding sites at its 3'UTR region (see Fig 1C). More importantly, EIDl has been shown to have a close relationship with two essential hematopoiesis-related transcriptional coactivators (S. Miyake et al., MoI Cell Biol. 20, 8889-902 (2000) ; W. Xu et al., Blood. 107, 4407-16 (2006).).
• CBP Cyclic adenosine monophosphate response element binding protein
• p300 interacts with over 312 proteins, at least 65 of which are encoded by genes that are essential for hematopoiesis
• CBP/p300 was thought to provide both an assembly platform as well as protein acetyltransferase functions with many transcription factors and histones that regulate gene expression (V. V.
• shR-CH mesenchymal stem cells from human bone marrow are infected with those vectors.
• FIG. 2B the levels of shR-CH were significantly elevated by transduction of shR-CH vectors, compared with those in control or mock infected cells. Elevated levels of shR-CH can be sustained for a long time in BMSCs infected with shR-CH (data not shown).
• Levels of EIDl mRNA were analyzed via application of RT-PCR at day 2 following transduction of shR-CH vectors.
• BMSCs and HSCs it was noted that a reciprocal relationship between the expression of EIDl and the expression of the CD45 marker that is characteristic for hematopoietic cells. Moreover, CD45 was upregulated after shR-CH induced repression of EIDl, raising the possibility that EIDl is involved in suppressing the hematopoietic program in BMSCs. According to the present invention a single BMSC was cloned by the single cell cloning method to reach this end and rule out the possibility of HSC contamination.
• results from a fluorescence activated cell sorter illustrated that the cloned BMSCs were characterized with the presence of specific CD29, CD 105 and CD44 surface antigens and FIk-I nuclear antigen, and without the CD34, CD45 and CD133 specific for HSCs (Suppl. Fig. 1). Then these cloned cells were infected with vectors expressing shR-CH or mock shRNA and seeded onto the 24-well plate coated with f ⁇ bronectin and collagen, and supplemented with a cocktail of hematopoietic cytokines and growth factors.
• BMSCs originating from transduced BMSCs were identified on the basis of the green fluorescent protein (GFP) marker carried by the vector, and differentiation of BMSCs to hematopoietic cells was characterized by expression of specific CD45 surface antigen (Fig. 3A).
• GFP green fluorescent protein
• Fig. 3A Ex vivo expansion of infected BMSCs was determined by using a modified CFU assay. 5000 seed cells from each group were selected and cultured for an additional 14 days.
• shR-CH overexpression rendered a substantial growth advantage during the 14-day culture as determined by in vitro colony-forming capacity (CFU-GM). This was also reflected by the predominance of GFP- and CD45- positive cells in the shR-CH culture (Fig. 3B).
• si-EIDl is highly specific for its target mRNA since mock siRNAs showed no effects on the differentiation of BMSCs. Therefore, like the ectopic expression of shR-CH, the knockdown of EIDl expression in BMSCs can result in the differentiation of these stem cells toward hematopoietic cells. Together, the findings described above provide the direct evidence that shR-CH dictates the differentiation of MSCs via the repression of EIDl.
• the cells were analyzed by flow cytometry by using three monoclonal antibodies against human leukocyte and stem cell markers. Consistent with the observation on the bone marrow sections, FACS profiles of bone marrow cells from recipients also showed robust engraftment of the BMSC-derived cells in medullary canal (Fig. 4B). After 8 weeks, the lineage composition of medullary canal cells descending from infected human BMSC (GFP + cells) was examined. The shR-CH expression in human BMSCs led to a significant increase in lymphoid (CD3) cells in medullary canal (2.8% vs 0.5% in the control).
• CD3 lymphoid
• Fig. 4B there was also a substantial elevation in CD33-positive myeloid lineage cells.
• shR-CH The preferential expression of shR-CH in HSCs implicates an important role of shR-CH in hematopoiesis.
• the findings in the invention show that ectopic expression of shR-CH is able to initiate and direct the human BMSCs to differentiate into hematopoietic cell lines in vitro and vivo (Fig. 3 and 4) substantiate that notion.
• EIDl EIDl
• shR-CH probably up-regulates the activities of hematopoiesis-related genes such as CBP and p300 (Fig 4C).
• CBP and p300 knockout or point-mutant mice were reported to have dramatically reduced numbers of definitive erythroid, myeloid, and B-lymphocytic progenitors in bone marrow (L. H. Kasper, Nature. 419, 738-43 (2002); Y. Chen, P. Haviernik, K.D. Bunting, Y.C. Yang, Blood. 110, 2889-98 (2007); A.L. Kung et al, Genes Dev. 14, 272-7 (2000)).
• Other studies further showed that CBP and p300 were fate decision factors for HSCs, responsible for HSC self-renewal and hematopoietic differentiation, respectively (V.I. Rebel et al., Proc Natl Acad Sci U S A.
• Figure 1 shows an identification and characterization of a novel shR-CH in human stem cells, wherein Figure IA indicates the differential expression of newly identified shR-CH. Data are averages of at least three independent determinations. Error bars indicate standard deviations. *P ⁇ 0.01 and **P ⁇ 0.001, expression of shR-CH detected by microarrays or qRT-PCR assay in BMSCs compared with HSCs, respectively.
• Figure IB indicates the cloning and sequencing of mature shR-CH and the secondary structure of the same. Solid red line stands for the sequence of mature shR-CH.
• Figure 1C indicates the alignment of the sequences of mature shR-CH and its three putative binding sites at the 3'UTR region of EIDl mRNA.
• FIG. 2 shows that the ectopic expression of shR-CH can effectively inhibit the expression of endogenous EIDl gene at the translational level.
• Fig 2A indicates the construction of the mock-sRNA /GFP and shR-CH/GFP retroviral vectors used in this study.
• Fig 2B indicates the differential expression of newly identified shR-CH in different cases. Data are averages of at least three independent determinations. Error bars indicate standard deviations. *p ⁇ 0.001.
• Fig 2C indicates Real Time-PCR analysis on the expression levels of EIDl mRNA under various conditions were carried out and normalized to that of GAPDH, and the resultant expression levels in different cases are normalized to their levels in the control. Data are averages of at least three independent determinations. Error bars indicate standard deviations.
• Fig 2D indicates the levels of EIDl protein from different cases were analyzed by Western blotting. The Western blot was stripped and re-probed with actin antibody to check for equal loading of total protein.
• Figure 3 shows that the enforced expression of shR-CH can induce the proliferation and differentiation of human BMSCs in vitro.
• Cells from a single clone were used.
• Fig 3A indicates the morphological alterations of normal BMSC, mock-, shR-CH- and siEIDl -infected BMSCs illustrated by different staining methods.
• Fig 3B indicates CFC assay of CD45 + hematopoietic cells. Cells were cultured in growth medium for 24 hours after transduction and then transferred into differentiation medium for 12 hours before immunostaining for CD45 specific for hematopoietic cells.
• Fig 3C indicates quantitative analysis of colonies from three different cases. At Day 14, the colony number from the BMSCs infected by shR-CH, siEIDl or mock vectors gave the result of n>6/case; *p ⁇ 0.001. Value inside bars represents fold increase.
• Figure 4 shows the effects of shR-CH forced expression of on hematopoietic lineage differentiation in vivo, wherein Fig 4A indicates the immunofluorescent and Hochest33342 staining of mouse bone marrow sections at 60 days post-trans. Enumeration of cells were stained with the human- specific antibodies against CD45, CD3 or CD33 in control, mock-shRNA and shR-CH sections of bone marrow obtained at 60 days post-transplantation.
• Fig 4B indicates hematopoietic reconstitution from human BMSCs grafted in NOD-SCID mice. 10 5 GFP + CD45 + BMSCs were transplanted in sublethally irradiated NOD-SCID mice.
• Fig 4C and Fig 4D indicate that Real time -PCR analysis on the expression levels of RUNXl mRNA under various conditions were carried out and normalized to that of GAPDH, and the resultant expression levels in different cases are normalized to their levels in the control. Data are averages of at least three independent determinations. Error bars indicate standard deviations.
• BM Human bone marrow
• mononuclear cells were separated by a Ficoll-Paque gradient centrifugation (specific gravity 1.077 g/mL; Nycomed Pharma AS, Oslo, Norway) and cultured in DF12 medium containing 5% FCS, 20 ng/niL EGF, 100 U/mL penicillin and 100 g/mL streptomycin (Gibco Life Technologies) at 37°C and a 5%CO 2 humidified atmosphere. The floating cells were discarded at 18 hours. Culture media were changed every 2 days.
• cells were harvested and further depleted of hematopoietic cells with magnetic-activated cell separation (MACS) CD45, GIyA, and CD34 micromagnetic beads (Miltenyi Biotec, Auburn, CA). Cells were then replated and passaged. At passages 4 cells were harvested by trypsinization, transfected with retrovirus vectors and used in the transplantation assay. Cells derived from single clone were used in other assays. To ensure single-cell originality of each cell colony, sorted cells were plated in wells coated with fibronectin (Sigma, St Louis, MO) and collagen (Sigma) for each patient.
• fibronectin Sigma, St Louis, MO
• collagen Sigma
• Culture medium was Dulbecco modified Eagle medium and Ham F 12 medium (DF 12) containing 40% MCDB-201 medium complete with trace elements (MCDB) (Sigma), 2% fetal calf serum (FCS; Gibco Life Technologies, Paisley, United Kingdom), 1: insulin-transferrin-selenium (Gibco Life Technologies), 10 - " 9 M dexamethasone (Sigma), 10 ⁇ 4 M ascorbic acid 2-phosphate (Sigma), 20 ng/mL interleukin-6 (Sigma), 10 ng/mL epidermal growth factor (Sigma), 10 ng/mL platelet-derived growth factor BB (Sigma), 50 ng/mL fetal liver tyrosine kinase 3 (Flt-3) ligand (Sigma), 30 ng/mL bone morphogenetic protein-4 (Sigma), and 100 LVmL penicillin and 100 g/mL streptomycin.
• MCDB 2% fetal calf serum
• a retrovirus vector pMSCV encoding shRNAs expressed from the U6 promoter was generated by the method in the art (Michael T Hemann 2003)., the short hairpin DNA sequence (SEQ. ID. NO.: 3 and SEQ. ID. NO.: 4, Table 4) coincident with the pre-shR-CH was chemically synthesized as a construct.
• the retroviruses containing the shR-CH-expressing cassette were packaged by H293T cells.
• the retroviral titers of the mock-GFP and shR-CH-GFP producer cells were 3 x 105/mL and 4 x 105/mL respectively, as assessed by transfer of GFP expression to H293T cells. (Fig.2 A)
• Short hairpin RNAs were polyadenylated at 37 ° C for 30 min in a 50-ul reaction volume including 1.5 ug RNA and 5 U poly(A) polymerase (Takara). Poly(A)-tailed small RNA was recovered by phenol/chloroform extraction and ethanol precipitation. Reverse transcription was performed by applying 1.5 ug RNA and 1 ug of RT primer (SEQ. ID. NO.: 5, Table 4) with 200 U of Superscript III reverse-transcriptase (Invitrogen). The cDNA amplification was carried out for 40 cycles at a final annealing temperature of 60 ° C by using primers SEQ. ID. NO.: 6 (Table 4) targeting specifically the novel shRNA shR-CH and SEQ.
• a 5' adapter (SEQ. ID. NO.: 8, Table 4) was ligated to small RNA by using T4 RNA ligase (TAKARA) and the ligation products were recovered by phenol/chloroform extraction followed by ethanol precipitation. Reverse transcription was performed by means of 1.5 ug RNA and 1 ug specific RT primer, SEQ. ID. NO.: 9 (Table 4). The cDNA amplification was carried out for 40 cycles at a final annealing temperature of 60 ° C via primers, SEQ. ID. NO.: 10 and SEQ. ID. NO.: 11 (Table 4). The PCR products were separated on 2% agarose with Goldview staining.
• RNA and related genes were determined by real-time RT-PCR.
• SYBR green assays with SYBR Premix Ex TaqTM (TAKARA) were run on the 7500HT real-time PCR instrument (Applied Biosystems).
• Reverse transcription of small RNA was performed by using 1.5 ug RNA and 1 ug shR-CH specific RT primer .
• beta-actin was used as loading control.
• the antibodies used included beta-actin (Santa Cruz Biotechnology), and EIDl (UPSTATE).
• the membranes then were incubated for one hour with HRP-conjugated rabbit anti-goat secondary antibody. Immunocomplexes were visualized with a commercial ECL kit.(Fig.2D) Example 7 Bioinformatic Analysis
• miRanda 3.0 and targetscan 3.1 ( Miranda KC et al., Cell. 126, 1203-1217 (2006); Grimson A, Farh KK et al., MoI CeI. 27, 91-105 (2007)), were used in the invention for target prediction for shRNA.
• HSCs-overexpressed human-shR-CH was selected for target verification.
• one potential targets for human shR-CH, EIDl was chosen for further experimental validation. The result was shown in Fig.1 C .
• CFU-GM Granulocyte-macrophage colony-forming unit
• Cells from a single clone were fixed with paraformaldehyde 4% for 30 min at room temperature and then washed three times with PBS. The cells were permeabilized with Triton 0.2% in PBS for 5 min. After four washes in PBS, the cells were blocked with a blocking buffer (Dako) for 30 min and then incubated in PBS/BSA 0.1% with an anti-albumin (Dako) or anti-CD45, CD34, CD19, CD71, CD3 and CD33 antibody (PE; Becton Dickinson), or with rabbit IgG (Jackson ImmunoResearch) as a negative control for one hour at room temperature.
• Dako anti-albumin
• PE Becton Dickinson
• rabbit IgG Jackson ImmunoResearch
• the cells were washed three times in PBS and incubated with an antirabbit whole IgG-Cy3 (Jackson ImmunoResearch) or anti-rabbit whole IgG-Cy2 (Jackson ImmunoResearch) in PBS/BSA 0.1% for one hour at room temperature.
• the cells were washed again (four times in PBS) and the nuclei were stained with Hochest 33342 diluted in PBS for 5 min at room temperature.
• the stained cells were visualized by using a fluorescence microscope (Leica DMIRB) and images captured using Magnaf ⁇ re software. The results refer to Fig.3A, Fig.4A.
• mice Six to eight week-old male nonobese diabetic (NOD)/ LtSz-scid/scid (SCID ) mice were bred and maintained under defined flora conditions in individually ventilated (high-efficiency particle-arresting filtered air) sterile micro-isolator cages (Techniplast, Milan, Italy). All animal handle and experiment procedures were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences. Mice were sublethally irradiated (300 cGy) with a cesium source (MDS Nordion; Gammacell, Ottawa, QC, Canada) prior to transplantation.
• NOD nonobese diabetic
• SCID LtSz-scid/scid mice
• BMSCs 2.5 ⁇ l O 5 cells/mice transduced without or with shR-CH vectors or mock vectors in 0.4 ml of physiological saline (PS) were respectively injected via tail vain into the irradiated mice.
• PS physiological saline
• the peripheral white blood cell count was done once a week. Mice were killed 2 months later by cervical dislocation.
• the BM from both femora and tibiae were collected. They were air dried and then stained with Jenner-Giemsa (BDH Ltd, Poole, United Kingdom). Conventional 4-um histologic sections of decalcified tibia were cut from formalin-fixed, paraffin-embedded material and stained.
• mice that received a transplant was assessed for presence of human cells by FACS.
• Mononuclear cells were harvested as described in the previous paragraph and red blood cells were lysed by adding 8.3% ammonium chloride. Single-cell suspensions were then prepared. After blocking of Fc receptors with human serum, cells were determined by labeling with anti-CD45, -CD33, or -CD3 phycoerythrin (PE; Becton Dickinson). Cells labeled with anti-immunoglobulin G (anti-IgG) monoclonal antibody (mAb) were used as control. In addition, cells were gated to include both lymphoid and myeloid fractions. (Fig.4B)

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