cDNA Representational Difference Analysis Research Articles

Next-generation sequencing of representational difference analysis products for identification of genes involved in diosgenin biosynthesis in fenugreek (Trigonella foenum-graecum)

Main conclusionRepresentational difference analysis of cDNA was performed and differential products were sequenced and annotated. Candidate genes involved in biosynthesis of diosgenin in fenugreek were identified. Detailed mechanism of diosgenin synthesis was proposed.Fenugreek (Trigonella foenum-graecum L.) is a valuable medicinal and crop plant. It belongs to Fabaceae family and has a unique potential to synthesize valuable steroidal saponins, e.g., diosgenin. Elicitation (methyl jasmonate) and precursor feeding (cholesterol and squalene) were used to enhance the content of sterols and steroidal sapogenins in in vitro grown plants for representational difference analysis of cDNA (cDNA-RDA). To identify candidate genes involved in diosgenin biosynthesis, differential, factor-specific libraries were subject to the next-generation sequencing. Approximately 9.9 million reads were obtained, trimmed, and assembled into 31,491 unigenes with an average length of 291 bp. Then, functional annotation and gene ontogeny enrichment analysis was performed by aligning all-unigenes with public databases. Within the transcripts related to sterol and steroidal saponin biosynthesis, we discovered novel candidate genes of diosgenin biosynthesis and validated their expression using quantitative RT-PCR analysis. Based on these findings, we supported the idea that diosgenin is biosynthesized from cycloartenol via cholesterol. This is the first report on the next-generation sequencing of cDNA-RDA products. Analysis of the transcriptomes enriched in low copy sequences contributed substantially to our understanding of the biochemical pathways of steroid synthesis in fenugreek.

Detailed Molecular Characterisation of the Transgenic Potato Line, AppA6, Modified with the Apple (Malus domestica) Polygalacturonase Inhibiting Protein 1 (pgip1) Gene

Current safety assessment of genetically modified crops requires detailed information about the insertion of the transgene and the effect of its expression on the biochemistry and physiology of the host plant. Whilst the intended effect of the transformation can be verified through phenotypic screening, molecular approaches are required to observe unintended effects. We investigated the molecular details of the integration of a polygalacturonase inhibiting protein 1 gene from Malus domestica (Mdpgip1), overexpressed in Solanum tuberosum (cv BP1) for enhanced resistance against Verticillium wilt. Genome walking studies of the selected AppA6 transformant revealed that the T-DNA containing the Mdpgip1 transgene under control of the CaMV 35S promoter was inserted into the genome without any non-T-DNA sequences from the pCAMBIA2300 vector. Sequence data indicate that the insertion of the Mdpgip1 transgene was in a gene-rich region of chromosome 1, adjacent to the photosystem Q B gene but without disruption of structural genes. Transcriptome-based cDNA-representational difference analysis revealed the distinctive expression of Mdpgip1 in the transgenic AppA6 line, verifying the intended effect. Protein extracts from the transgenic plants inhibited the activities of Verticillium dahliae polygalacturonases in in vitro studies, showing that the transgene is expressed to produce an active PGIP defense protein. cDNA-AFLP fingerprinting revealed genes that were differentially expressed, including genes encoding tryptophan/tyrosine permease, Ef-Tu domain and SKP1-like 1A proteins. qRT-PCR indicated that the Mdpgip1 transgene insertion resulted in increased expression in the AppA6 transgenic of the xyloglucan endotransglycosylase (xth) gene and an endogenous Stpgip1 gene. These unintended changes were either caused by the constitutive expression of the Mdpgip1 transgene or transformation-related somaclonal variation. The results indicate that the stable, single copy integration of the Mdpgip1 gene in the AppA6 transgenic line did not disrupt any structural genes but caused unintended effects that affected gene expression compared to the parental counterpart under the non-stressed experimental conditions investigated.

Evolution of the strategies for screening and identifying human tumor antigens.

The prevailing view is that not only can some of the tumor antigens be used as biosensors for cancers, but also they may indeed be used as targets for immunotherapy. The identification of tumor antigens becomes a vital step in oncology research. Both the humoral immune system and the cellular immune system are activated in response to a tumor antigen in vivo of patients with tumor. Immune effector molecules and cells can be used to screen and identify tumor antigens. Specific T cells, including CD8(+) and CD4(+) T cells, can identify T cell epitopes, and specific antibodies in sera can identify B cell epitopes. The researchers have studied this area for decades. Initially, they explored tumor antigens with the use of 1-D SDS-PAGE and sandwich ELISAs. Since 1990s, CTL screening approach and peptide elution approach had been established. After that, SEREX, SERPA and protein microarray technology have become the mainstream highthroughput strategies for identifying tumor antigens. There are some other approaches, such as combinatorial peptide libraries, representational difference analysis of cDNA and bioinformatics methods. This review's aim is to describe the generation, the theory, the key protocols and the application of some main techniques and provide their benefits and drawbacks.

Reduced tolerance to abiotic stress in transgenic Arabidopsis overexpressing a Capsicum annuum multiprotein bridging factor 1.

BackgroundThe pepper fruit is the second most consumed vegetable worldwide. However, low temperature affects the vegetative development and reproduction of the pepper, resulting in economic losses. To identify cold-related genes regulated by abscisic acid (ABA) in pepper seedlings, cDNA representational difference analysis was previously performed using a suppression subtractive hybridization method. One of the genes cloned from the subtraction was homologous to Solanum tuberosum MBF1 (StMBF1) encoding the coactivator multiprotein bridging factor 1. Here, we have characterized this StMBF1 homolog (named CaMBF1) from Capsicum annuum and investigated its role in abiotic stress tolerance.ResultsTissue expression profile analysis using quantitative RT-PCR showed that CaMBF1 was expressed in all tested tissues, and high-level expression was detected in the flowers and seeds. The expression of CaMBF1 in pepper seedlings was dramatically suppressed by exogenously supplied salicylic acid, high salt, osmotic and heavy metal stresses. Constitutive overexpression of CaMBF1 in Arabidopsis aggravated the visible symptoms of leaf damage and the electrolyte leakage of cell damage caused by cold stress in seedlings. Furthermore, the expression of RD29A, ERD15, KIN1, and RD22 in the transgenic plants was lower than that in the wild-type plants. On the other hand, seed germination, cotyledon greening and lateral root formation were more severely influenced by salt stress in transgenic lines compared with wild-type plants, indicating that CaMBF1-overexpressing Arabidopsis plants were hypersensitive to salt stress.ConclusionsOverexpression of CaMBF1 in Arabidopsis displayed reduced tolerance to cold and high salt stress during seed germination and post-germination stages. CaMBF1 transgenic Arabidopsis may reduce stress tolerance by downregulating stress-responsive genes to aggravate the leaf damage caused by cold stress. CaMBF1 may be useful for genetic engineering of novel pepper cultivars in the future.

Identification of endometriosis‐related genes by representational difference analysis of cDNA

Accumulated evidence reveals that abnormally expressed genes in eutopic endometrium of endometriosis play a critical role in the pathogenesis of endometriosis. Identification of endometriosis-related genes for further revealing the pathogenesis of endometriosis and offering the basis for developing the molecular-targeted diagnosis and therapy of endometriosis. Forty women with endometriosis and forty control women without endometriosis during their secretory phase were selected for this study. cDNA representational difference analysis (cDNA-RDA) was performed to screen the up-regulated genes in eutopic endometrium samples of endometriosis (n = 10) compared with the controls (n = 10). To validate the results, MAT2A, the most abundantly expressed gene, was selected to detect mRNA and protein levels between eutopic endometrium of endometriosis (n = 40) and controls (n = 40) using immunohistochemistry, real-time fluorescent quantitative PCR and Western blotting. Ten up-regulated genes were identified in eutopic endometria of endometriosis compared with controls. Among these genes, COX-2, BRAF, NRAS and CFL1 have already been reported to be associated with the endometriosis in previous studies. MAT2A, SEPT9, ATAD3A and CADM2 have been reported to be involved in other diseases but not in endometriosis. NAA15 and CCDC21 have not reported in any diseases. Further study showed that MAT2A protein was localised in both endometrial glandular and stromal cells. Compared with controls, the mRNA and protein levels of MAT2A were significantly higher in eutopic endometrium of endometriosis (P < 0.05). cDNA-RDA can be used to effectively identify the endometriosis-related genes, which can provide novel experimental data and further understand the pathogenesis of endometriosis.

CDNA representational difference analysis for identifying transcripts regulated under anhydrobiosis in the tardigrade Milnesium tardigradum

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In vitro requirement for periostin in B lymphopoiesis

AbstractB lymphopoiesis arrests in rabbits by 4 months of age. To identify molecules that contribute to this arrest, cDNA–representational difference analysis on BM stromal cells from young and adult rabbits showed that expression of Postn that encodes for the extracellular matrix protein periostin dramatically reduced with age. Postn–small interfering RNA OP9 cells lost their capacity to support B-cell development from rabbit or murine BM cells, and reexpression of periostin restored this potential, indicating an in vitro requirement for periostin in B lymphopoiesis. In our system, we determined that periostin deficiency leads to increased cell death and decreased proliferation of B-lineage progenitors. Further, RGD peptide inhibition of periostin/αvβ3 interaction resulted in a marked decrease in B lymphopoiesis in vitro. Microarray analysis of the Postn–small interfering RNA OP9 cells showed decreased expression of key B-lymphopoietic factors, including IL-7 and CXCL12. In vivo, unidentified molecule(s) probably compensate periostin loss because Postn−/− mice had normal numbers of B-cell progenitors in BM. We conclude that the decline in periostin expression in adult rabbit BM does not solely explain the arrest of B lymphopoiesis. However, the interaction of periostin with αvβ3 on lymphoid progenitors probably provides both proliferative and survival signals for cells in the B-cell development pathway.

Cloning and stage-specific expression of CK-M1 gene during metamorphosis of Japanese flounder, Paralichthys olivaceus

The symmetrical body of flatfish larvae changes dramatically into an asymmetrical form after metamorphosis. The molecular mechanisms responsible for this change are poorly understood. As an initial step to clarify these mechanisms, we used representational difference analysis of cDNA for the identification of genes active during metamorphosis in the Japanese flounder, Paralichthys olicaceus. One of the up-regulated genes was identified as creatine kinase muscle type 1 (CK-M1). Sequence analysis of CK-M1 revealed that it spanned 1 708 bp and encoded a protein of 382 amino acids. The overall amino acid sequence of the CK-M1 was highly conserved with those of other organisms. CK-M1 was expressed in adult fish tissues, including skeletal muscle, intestine and gill. Whole mount in-situ hybridization showed that the enhanced expression of CK-M1 expanded from the head to the whole body of larvae as metamorphosis progressed. Quantitative analysis revealed stage-specific high expression of CK-M1 during metamorphosis. The expression level of CK-M1 increased initially and peaked at metamorphosis, decreased afterward, and finally returned to the pre-metamorphosis level. This stage-specific expression pattern suggested strongly that CK-M1 was related to metamorphosis in the Japanese flounder. Its specific role in metamorphosis requires further study.

Differentially expressed genes under simulated microgravity in fruiting bodies of the fungus Pleurotus ostreatus

In response to a change in the direction of gravity, morphogenetic changes of fruiting bodies of fungi are usually observed as gravitropism. Although gravitropism in higher fungi has been studied for over 100 years, there is no convincing evidence regarding the graviperception mechanism in mushrooms. To understand gravitropism in mushrooms, we isolated differentially expressed genes in Pleurotus ostreatus (oyster mushroom) fruiting bodies developed under three-dimensional clinostat-simulated microgravity. Subtractive hybridization, cDNA representational difference analysis was used for gene analysis and resulted in the isolation of 36 individual genes (17 upregulated and 19 downregulated) under clinorotation. The phenotype of fruiting bodies developed under simulated microgravity vividly depicted the gravitropism in mushrooms. Our results suggest that the differentially expressed genes responding to gravitational change are involved in several potential cellular mechanisms during fruiting body formation of P. ostreatus.

Cell adaptation to activated FGFR3 includes Sprouty4 up regulation to inhibit the receptor-mediated ERKs activation from the endoplasmic reticulum

The kinase activity of the thanatophoric dysplasia type II–fibroblast growth factor receptor 3 mutant (TDII–FGFR3) hampers its maturation. As a consequence, the immature receptor activates extracellular regulated kinases (ERKs) from the endoplasmic reticulum (ER), which leads to apoptosis. On the other hand, in stable TDII–FGFR3 cells receptor biosynthesis is restored and ERKs are activated from the cell surface. To identify potential mediators of cell adaptation to the activated receptor we investigated gene products that are differently regulated in TDII and wild-type FGFR3 cells. cDNA representational difference analysis reveals Sprouty4 up regulation in the TDII–FGFR3 cells. Interestingly, Sprouty4 inhibits the TDII–FGFR3-mediated ERKs activation from the ER, but fails to suppress ERKs activation from cell surface. We conclude that cell adaptation to activated FGFR3 include Sprouty4 activity, which silences the premature receptor signaling and suppress apoptosis.

Downregulation of ubiquitin gene expression in the deltamethrin‐resistant diamondback moth (Plutella xylostella L.)

Abstract Plutella xylostella (L.) has long been regarded as a very important universal pest of cruciferous crops. It is therefore important to understand its mechanisms of insecticide resistance. In our previous study, we obtained a differently expressed sequence with relatively high homology to the ubiquitin (UBI) gene as assessed via cDNA representational difference analysis (RDA). To further study this product, we cloned UBI open reading frame (ORF) sequences identified from both sexes of the deltamethrin‐resistant strain (DRF and DRM) and the deltamethrin‐sensitive strain (SF and SM) of P. xylostella. The deduced protein sequence of DRM was different from the other samples. The expression level was measured using real‐time quantitative polymerase chain reaction (PCR) and Western blot. The results of both analyses revealed the expression levels, in order from highest to lowest, to be in SF, SM, DRF and DRM. This indicates that the long‐term use of deltamethrin on P. xylostella results in decreased expression of the UBI gene.

Screening of eye-position related genes with DD-RT-PCR and RDA in the hybrids between Japanese flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus

Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae were screened with classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA (cDNA-RDA); 30 and 47 putative fragments were isolated, respectively. The cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited eye-position expression patterns during metamorphosis. However, none of the fragments was proved to be related to flatfishes’ eye-position specifically. Therefore, further studies and more sensitive gene isolated methods are needed to solve the problems.

Open Architecture PCR-Based Methods for Differential Gene Expression Analysis

Identification and isolation of differentially expressed genes is of great interest for a broad range of medical and biological research fields. For pharmaceutical and pharmacological applications the identification of target genes is of importance as well as the investigation of the transcriptional response to drug administration. Over the last two decades several techniques have been developed for analyzing gene expression in a variety of experimental contexts. Whereas microarrays represent closed architecture systems, restricted to well characterized models with a vast amount of available background data (e.g. human, mouse, etc.), open architecture systems allow detection of novel genes without a priori knowledge of the transcriptome. They are often based on amplification, subtraction and comparative display, and are well suited for so-called non-model organisms with little or no sequence data available. We review here the most common methods and their modifications: mRNA fingerprinting, differential display (DD), cDNA representational difference analysis (RDA) and suppression subtractive hybridization (SSH). The focus is on PCR-based methods that can be conducted with minimal bioinformatic and/or high throughput facility support, making them applicable in virtually any laboratory having access to basic molecular biology equipment. Furthermore we will give an insight into applications of these techniques in the pharmaceutical / pharmacological field. Keywords: Open architecture systems, SSH, cDNA RDA, mRNA fingerprinting, Differential gene expression analysis

Selection of serotype-specific vaccine candidate genes in Actinobacillus pleuropneumoniae and heterologous immunization with Propionibacterium acnes

Actinobacillus pleuropneumoniae ( A. pleuropneumoniae) is a highly contagious lethal causative agent of swine pleuropneumoniae. Vaccines for this disease are usually serotype specific. In order to identify immunogenic genes specific to serotypes, two differentially expressed gene cDNA libraries of A. pleuropneumoniae CCVC259 (serotype 1) and CCVC263 (serotype 5) had been constructed by using a cDNA representational difference analysis (cDNA-RDA). From the libraries, six potential vaccine candidate genes expressed only in serotype 1 and 13 genes in serotype 5 were identified by antibody screening after gene expression in vitro with a ribosome display system. Eight sequences out of these exhibited 77–100% identity to the corresponding genes in Propionibacterium acnes. The antisera raised against A. pleuropneumoniae serotypes 1 and 5 were reactive with P. acnes at a titer of 1:6400 and vice versa (ELISA titer, 1:3200). Mice immunized with P. acnes were protected against 10 × LD 50 challenge with A. pleuropneumoniae serotypes 1 and 5, and the survival rates were 90% and 95%, respectively. Pigs vaccinated with the P. acnes strain could develop high level antibody cross-reacted with A. pleuropneumoniae and obtain noticeable protection from A. pleuropneumoniae infection. These data demonstrate that there were common antigens between A. pleuropneumoniae and P. acnes, and the cross protectivity highlights the possibility of using P. acnes vaccines for preventing infection by A. pleuropneumoniae.

Identification of novel and known ovary‐specific genes including ZP2, in a marsupial, the stripe‐faced dunnart

During the early stages of oogenesis, oocyte-specific factors, synthesized by and stored within the oocyte, play critical roles during oogenesis, folliculogenesis, fertilization and early embryonic development in the mouse. The identification of marsupial maternal factors, expressed specifically in the ovary or oocyte, may provide an insight into the conserved evolutionary mechanisms that drive mammalian oocyte development to cleavage stages. In this study, 10 clones including dunnart ZP2 and c-mos, isolated by cDNA representational difference analysis, were validated by RT-PCR for ovary-specific expression. This novel combination of techniques to isolate ovary-specific genes has identified three novel genes with ovary-specific expression. Both dunnart ZP2 and c-mos exhibited ovary-specific expression, making this study the first isolation of c-mos in a marsupial species. Dunnart ZP2 expression was examined in detail by in situ hybridization and results indicate oocyte-specific expression of dunnart ZP2 in the cytoplasm of oocytes of primordial, primary and secondary follicles with expression being highest in oocytes of primary follicles. ZP2 was not expressed in granulosa cells of any follicles.

CDNA representational difference analysis used in the identification of genes expressed by Trichophyton rubrum during contact with keratin

Dermatophytes are adapted to infect skin, hair and nails by their ability to utilize keratin as a nutrient source. Trichophyton rubrum is an anthropophilic fungus, causing up to 90% of chronic cases of dermatophytosis. The understanding of the complex interactions between the fungus and its host should include the identification of genes expressed during infection. To identify the genes involved in the infection process, representational difference analysis (RDA) was applied to two cDNA populations from T. rubrum, one transcribed from the RNA of fungus cultured in the presence of keratin and the other from RNA generated during fungal growth in minimal medium. The analysis identified differentially expressed transcripts. Genes related to signal transduction, membrane protein, oxidative stress response, and some putative virulence factors were up-regulated during the contact of the fungus with keratin. The expression patterns of these genes were also verified by real-time PCR, in conidia of T. rubrum infecting primarily cultured human keratinocytes in vitro, revealing their potential role in the infective process. A better understanding of this interaction will contribute significantly to our knowledge of the process of dermatophyte infection.

Screen for genes in periaqueductal grey of male Wistar rats related to reduced exploratory activity in the elevated plus-maze

Aim of a present study was to find genes in the periaqueductal grey (PAG) related to the exploratory behavior in rats. Male Wistar rats were divided according to their exploratory behavior in the elevated plus-maze model of anxiety into two groups: high (non-anxious) and low (anxious) exploratory activity. Differential expression of genes was analyzed using the cDNA representational difference analysis (RDA). Q-RT-PCR was used to confirm most prominent changes and functional annotation of the identified genes was performed to establish pathways related to exploratory behavior of rats. We found different genetic activation related to the exploratory activity of rats. Rats with low exploratory activity showed increase in the intracellular signal transduction and in GABA, vasopressin and adrenergic receptor activities. Functional annotation confirmed significant induction of cAMP system and GTPases in rats with anxious-type behavior. On the other hand, rats with high exploratory activity in the elevated plus-maze (non-anxious type of behavior) had increased activity of genes forming “behavioral fear response” system. These changes were specific to PAG, because they were not found in the cerebellum. In addition, plasma corticosterone levels were significantly higher in rats with non-anxious behavior compared to anxious behavior. Our results show that non-anxious behavior is related to activation of “fear response system” and more intense activation of HPA axis. Possibly it means that this system helps animals to cope with the threatening circumstances. More detailed analysis of this potential “fear response system” is necessary in the further studies for understanding its role in the regulation of emotional behavior.

The fruiting-specific Le.flp1 gene, encoding a novel fungal fasciclin-like protein, of the basidiomycetous mushroom Lentinula edodes

To understand the molecular mechanisms of fruiting body formation of basidiomycetous mushrooms, we have isolated over a 100 of developmentally regulated genes that were specifically transcribed during fruiting body development in Lentinula edodes (Shiitake-mushroom) by a subtractive hybridization, cDNA-RDA (cDNA representational difference analysis). One of these genes, named Le.flp1, was isolated from the primordial cDNA library of L. edodes, and the expression product of Le.flp1 and putative fungal homologues contained a characteristic region, homologous to the Fas domain of fasciclin family proteins, which are capable of promoting cell adhesion through Fas domain-mediated homophilic interactions in various organisms. RT-PCR analyses suggested that Le.flp1 was specifically expressed in primordia and mature fruiting bodies. In situ hybridization indicated that Le.flp1 transcripts were distributed distinctly in the following tissues: the inside of gills of fruiting bodies, especially at the boundary between the subhymenium and trama, where there is active proliferation of basidium cells for producing basidiospores; peripheral regions of the primordium, pileus and stipe; and both inner tissue and outer regions of the stipe. Our results suggest the hypothesis that Le.flp1 plays a role in cellular differentiation and development in ubiquitous tissues during fruiting body formation in L. edodes, possibly through cell adhesion.

Identification of Genes Involved in Positive Selection of CD4+8+ Thymocytes: Expanding the Inventory

Positive selection of cortical CD4+8+ thymocytes represents crucial and mysterious process in T cell development whereby short-lived precursors are rescued from programmed cell death and induced to differentiate towards long-lived CD4 and CD8 T cells. One reason that this process is not fully understood is that the inventory of genes changing their expression in positively selected CD4+8+ thymocytes is not yet complete. In this work Affymetrics GeneChip cDNA microarrays and cDNA-Representational Difference Analysis were used to search for unknown and known genes that were not identified before as being involved in positive selection. Comparison of transcriptosome of nonstimulated with transcriptosome of PMA/ionomycin stimulated thymoma cell line resembling CD4+8+ thymocytes and subtraction of cDNA of extrathymic tissues from cDNA of purified CD4+8+ thymocytes resulted in identification of 36 genes, which have not been previously reported to change their expression during positive selection. One of them represents a novel, third evolutionarily conserved gene within RAG locus.

Penumbra Encodes a New Tetraspanin Critical to Effective Erythropoiesis.

To search for new genes that might be involved in the regulation of erythropoiesis, we performed cDNA representational difference analysis on a multipotent murine hematopoietic cell line, EML C1. Penumbra was identified based on its predominant, high-level expression in TER119+ erythroblasts. It is not expressed or is expressed at very low levels in Gr1+ or CD3+ or B220+ or CD11c or NK1.1+ bone marrow or spleen cells. The full-length murine Penumbra mRNA contains 2017 nucleotides and encodes a new membrane protein characterized by four transmembrane domains, a small extracellular domain between the first and second transmembrane domains and a large extracellular domain between the third and fourth transmembrane domains. It is a member of the evolutionarily conserved tetraspanin membrane protein superfamily that includes CD63, CD81, CD151 and Peripherin. Using the murine Penumbra as a probe, we identified the human Penumbra from a human bone marrow cDNA library. The human Penumbra also encodes a protein of 283 amino acids and is 97% identical to the murine Penumbra. The human Penumbra gene is mapped to chromosome 7q32, a hotspot for interstitial deletions in myelodysplastic syndromes and acute myelogenous leukemias. Immunofluorescence microscopy indicated that Penumbra is targeted to the cell surface. Immunoprecipitation-Western analysis showed that Penumbra formed disulfide bond-linked homodimers. To study the possible effects of Penumbra deletions, a knockout mouse model was created by homologous recombination. To avoid any pathology that might result from the Neo selectable marker, the targeting vector employed a self-excising Cre-Neo cassette driven by a testis-specific promoter. At 3 months of age, Penumbra−/− mice had similar numbers of RBC, hematocrits, WBC and platelets as the WT littermates. At 1 year of age, Penumbra−/− mice as a group had significantly lower RBC numbers and hematocrits than the WT littermates. Furthermore, about 40–60% of the Penumbra−/− mice had increased percentages of basophilic or polychromatophilic macrocytic RBC. As a group, these mice had even lower numbers of RBC (6.24 ± 1.87 vs. 8.88 ± 0.51 x 106/microliter in WT; p = 0.0002) and hematocrits (36.63 ± 8.28 vs. 46.21 ± 3.62% in WT; p = 0.002). Many such mice had massive splenomegaly due to increased extramedullary hematopoiesis. A multipotent hematopoietic cell line, EMX, was established from the bone marrow of a Penumbra−/−- mouse. EMX exhibits ineffective erythropoiesis in response to erythropoietin, a defect that is reversed by re-expression of Penumbra. These findings indicate that Penumbra plays an important role in erythropoiesis and suggest that Penumbra may be one of the genes that are often deleted during the evolution of myelodysplastic syndromes and acute myelogenous leukemias.