Perturbation of p38α MAPK as a Novel Strategy to Effectively Sensitize Chronic Myeloid Leukemia Cells to Therapeutic BCR-ABL Inhibitors.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by the presence of the BCR-ABL oncogene. Therapeutic regimens with tyrosine kinase inhibitors (TKIs) specifically targeting BCR-ABL have greatly improved overall survival of CML. However, drug intolerance and related toxicity remain. Combined therapy is effective in reducing drug magnitude while increasing therapeutic efficacy and, thus, lowers undesired adverse side effects. The p38 MAPK activity is critically linked to the pathogenesis of a number of diseases including hematopoietic diseases; however, the role of each isozyme in CML and TKI-mediated effects is still elusive. In this study, we used specific gene knockdown to clearly demonstrate that the deficiency of p38α greatly enhanced the therapeutic efficacy in growth suppression and cytotoxicity of TKIs, first-generation imatinib, and second generation dasatinib by approximately 2.5-3.0-fold in BCR-ABL-positive CML-derived leukemia K562 and KMB5 cells. Knockdown of p38ß, which displays the most sequence similarity to p38α, exerted distinct and opposite effects on the TKI-mediated therapeutic efficacy. These results show the importance of isotype-specific intervention in enhancing the therapeutic efficacy of TKI. A highly specific p38α inhibitor, TAK715, also significantly enhanced the imatinib- and dasatinib-mediated therapeutic efficacy, supporting the feasibility of p38α deficiency in future clinic application. Taken together, our results demonstrated that p38α is a promising target for combined therapy with BCR-ABL-targeting tyrosine kinase inhibitors for future application to increase therapeutic efficacy.

Arginine Methylation of hnRNPK Inhibits the DDX3-hnRNPK Interaction to Play an Anti-Apoptosis Role in Osteosarcoma Cells.

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA/DNA binding protein involved in diverse cell processes; it is also a p53 coregulator that initiates apoptosis under DNA damage conditions. However, the upregulation of hnRNPK is correlated with cancer transformation, progression, and migration, whereas the regulatory role of hnRNPK in cancer malignancy remains unclear. We previously showed that arginine methylation of hnRNPK attenuated the apoptosis of U2OS osteosarcoma cells under DNA damage conditions, whereas the replacement of endogenous hnRNPK with a methylation-defective mutant inversely enhanced apoptosis. The present study further revealed that an RNA helicase, DDX3, whose C-terminus preferentially binds to the unmethylated hnRNPK and could promote such apoptotic enhancement. Moreover, C-terminus-truncated DDX3 induced significantly less apoptosis than full-length DDX3. Notably, we also identified a small molecule that docks at the ATP-binding site of DDX3, promotes the DDX3-hnRNPK interaction, and induces further apoptosis. Overall, we have shown that the arginine methylation of hnRNPK suppresses the apoptosis of U2OS cells via interfering with DDX3-hnRNPK interaction. On the other hand, DDX3-hnRNPK interaction with a proapoptotic role may serve as a target for promoting apoptosis in osteosarcoma cells.

CDC25B induces cellular senescence and correlates with tumor suppression in a p53-dependent manner.

The phosphatase cell division cycle 25B (Cdc25B) regulates cell cycle progression. Increased Cdc25B levels are often detected in cancer cell lines and human cancers and have been implicated in contributing to tumor growth, potentially by providing cancer cells with the ability to bypass checkpoint controls. However, the specific mechanism by which increased Cdc25B impacts tumor progression is not clear. Here we analyzed The Cancer Genome Atlas (TCGA) database and found that patients with high CDC25B expression had the expected poor survival. However, we also found that high CDC25B expression had a p53-dependent tumor suppressive effect in lung cancer and possibly several other cancer types. Looking in more detail at the tumor suppressive function of Cdc25B, we found that increased Cdc25B expression caused inhibition of cell growth in human normal fibroblasts. This effect was not due to alteration of specific cell cycle stage or inhibition of apoptosis, nor by induction of the DNA damage response. Instead, increased CDC25B expression led cells into senescence. We also found that p53 was required to induce senescence, which might explain the p53-dependent tumor suppressive function of Cdc25B. Mechanistically, we found that the Cdc25B phosphatase activity was required to induce senescence. Further analysis also found that Cdc25B stabilized p53 through binding and dephosphorylating p53. Together, this study identified a tumor-suppressive function of Cdc25B that is mediated through a p53-dependent senescence pathway.

The MKK-Dependent Phosphorylation of p38α Is Augmented by Arginine Methylation on Arg49/Arg149 during Erythroid Differentiation.

The activation of p38 mitogen-activated protein kinases (MAPKs) through a phosphorylation cascade is the canonical mode of regulation. Here, we report a novel activation mechanism for p38α. We show that Arg49 and Arg149 of p38α are methylated by protein arginine methyltransferase 1 (PRMT1). The non-methylation mutations of Lys49/Lys149 abolish the promotive effect of p38α on erythroid differentiation. MAPK kinase 3 (MKK3) is identified as the major p38α upstream kinase and MKK3-mediated activation of the R49/149K mutant p38α is greatly reduced. This is due to a profound reduction in the interaction of p38α and MKK3. PRMT1 can enhance both the methylation level of p38α and its interaction with MKK3. However, the phosphorylation of p38α by MKK3 is not a prerequisite for methylation. MAPK-activated protein kinase 2 (MAPKAPK2) is identified as a p38α downstream effector in the PRMT1-mediated promotion of erythroid differentiation. The interaction of MAPKAPK2 with p38α is also significantly reduced in the R49/149K mutant. Together, this study unveils a novel regulatory mechanism of p38α activation via protein arginine methylation on R49/R149 by PRMT1, which impacts partner interaction and thus promotes erythroid differentiation. This study provides a new insight into the complexity of the regulation of the versatile p38α signaling and suggests new directions in intervening p38α signaling.

Calcium-dependent methylation by PRMT1 promotes erythroid differentiation through the p38α MAPK pathway.

Protein arginine methyltransferase 1 (PRMT1) stimulates erythroid differentiation, but the signaling events upstream are yet to be identified. Ca2+ plays crucial roles during erythroid differentiation. Here, we show that Ca2+ enhances methylation during induced erythroid differentiation and that Ca2+ directly upregulates the catalytic activity of recombinant PRMT1 by increasing Vmax toward the substrate heterogeneous nuclear ribonucleoprotein A2. We demonstrate that PRMT1 is essential and responsible for the effect of Ca2+ on differentiation. Depletion of Ca2+ suppresses PRMT1-mediated activation of p38α and p38α-stimulated differentiation. Furthermore, Ca2+ stimulates methylation of p38α by PRMT1. This study uncovers a novel regulatory mechanism for PRMT1 by Ca2+ and identifies the PRMT1/p38α axis as an intracellular mediator of Ca2+ signaling during erythroid differentiation.

Gene Expression Profiling and Pathway Network Analysis Predicts a Novel Antitumor Function for a Botanical-Derived Drug, PG2.

PG2 is a botanical drug that is mostly composed of Astragalus polysaccharides (APS). Its role in hematopoiesis and relieving cancer-related fatigue has recently been clinically investigated in cancer patients. However, systematic analyses of its functions are still limited. The aim of this study was to use microarray-based expression profiling to evaluate the quality and consistency of PG2 from three different product batches and to study biological mechanisms of PG2. An integrative molecular analysis approach has been designed to examine significant PG2-induced signatures in HL-60 leukemia cells. A quantitative analysis of gene expression signatures was conducted for PG2 by hierarchical clustering of correlation coefficients. The results showed that PG2 product batches were consistent and of high quality. These batches were also functionally equivalent to each other with regard to how they modulated the immune and hematopoietic systems. Within the PG2 signature, there were five genes associated with doxorubicin: IL-8, MDM4, BCL2, PRODH2, and BIRC5. Moreover, the combination of PG2 and doxorubicin had a synergistic effect on induced cell death in HL-60 cells. Together with the bioinformatics-based approach, gene expression profiling provided a quantitative measurement for the quality and consistency of herbal medicines and revealed new roles (e.g., immune modulation) for PG2 in cancer treatment.

Arginine methylation of hnRNPK negatively modulates apoptosis upon DNA damage through local regulation of phosphorylation.

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA/DNA-binding protein involved in chromatin remodeling, RNA processing and the DNA damage response. In addition, increased hnRNPK expression has been associated with tumor development and progression. A variety of post-translational modifications of hnRNPK have been identified and shown to regulate hnRNPK function, including phosphorylation, ubiquitination, sumoylation and methylation. However, the functional significance of hnRNPK arginine methylation remains unclear. In the present study, we demonstrated that the methylation of two essential arginines, Arg296 and Arg299, on hnRNPK inhibited a nearby Ser302 phosphorylation that was mediated through the pro-apoptotic kinase PKCδ. Notably, the engineered U2OS cells carrying an Arg296/Arg299 methylation-defective hnRNPK mutant exhibited increased apoptosis upon DNA damage. While such elevated apoptosis can be diminished through addition with wild-type hnRNPK, we further demonstrated that this increased apoptosis occurred through both intrinsic and extrinsic pathways and was p53 independent, at least in part. Here, we provide the first evidence that the arginine methylation of hnRNPK negatively regulates cell apoptosis through PKCδ-mediated signaling during DNA damage, which is essential for the anti-apoptotic role of hnRNPK in apoptosis and the evasion of apoptosis in cancer cells.

Elevation of protein kinase Cα stimulates osteogenic differentiation of mesenchymal stem cells through the TAT-mediated protein transduction system.

Mesenchymal stem cells (MSCs) can differentiate toward various lineages, including the osteogenic lineage, and thus hold great potential for clinic purposes. By using pharmacological inhibitors, protein kinase C (PKC) signaling has been shown to either negatively or positively regulate differentiation of bone, however, due to the low transfection efficiency in MSCs, the role of individual PKC isoforms is still not fully understood. In this study, we established a TAT peptide-mediated transduction system that efficiently delivered PKCα proteins into MSCs in a non-invasive fashion. The increased PKCα protein levels significantly promoted osteogenic differentiation in the murine mesenchymal C3H10T1/2 cells and in primary MSCs from both human and mouse, as demonstrated by the enhanced activity of the osteoblast marker, alkaline phosphatase, and the enhanced expression of the key transcription factor runx2. Mineralization is an important functional indication for bone differentiation. Our results further showed that PKCα promoted expression of the important osteocalcin gene and the accumulation of calcium minerals. Taken together, this study provides direct evidence showing that PKCα positively regulates osteogenic differentiation and demonstrates that the TAT peptide-mediated method enables functional study of specific PKC isoforms in MSCs without using viral infection. This may promote the application of PKCs in therapeutic treatment.

Protein arginine methyltransferase 1 interacts with and activates p38α to facilitate erythroid differentiation.

Protein arginine methylation is emerging as a pivotal posttranslational modification involved in regulating various cellular processes; however, its role in erythropoiesis is still elusive. Erythropoiesis generates circulating red blood cells which are vital for body activity. Deficiency in erythroid differentiation causes anemia which compromises the quality of life. Despite extensive studies, the molecular events regulating erythropoiesis are not fully understood. This study showed that the increase in protein arginine methyltransferase 1 (PRMT1) levels, via transfection or protein transduction, significantly promoted erythroid differentiation in the bipotent human K562 cell line as well as in human primary hematopoietic progenitor CD34(+) cells. PRMT1 expression enhanced the production of hemoglobin and the erythroid surface marker glycophorin A, and also up-regulated several key transcription factors, GATA1, NF-E2 and EKLF, which are critical for lineage-specific differentiation. The shRNA-mediated knockdown of PRMT1 suppressed erythroid differentiation. The methyltransferase activity-deficient PRMT1G80R mutant failed to stimulate differentiation, indicating the requirement of arginine methylation of target proteins. Our results further showed that a specific isoform of p38 MAPK, p38α, promoted erythroid differentiation, whereas p38ß did not play a role. The stimulation of erythroid differentiation by PRMT1 was diminished in p38α- but not p38ß-knockdown cells. PRMT1 appeared to act upstream of p38α, since expression of p38α still promoted erythroid differentiation in PRMT1-knockdown cells, and expression of PRMT1 enhanced the activation of p38 MAPK. Importantly, we showed for the first time that PRMT1 was associated with p38α in cells by co-immunoprecipitation and that PRMT1 directly methylated p38α in in vitro methylation assays. Taken together, our findings unveil a link between PRMT1 and p38α in regulating the erythroid differentiation program and provide evidence suggesting a novel regulatory mechanism for p38α through arginine methylation.

Proteomics analysis of in vitro protein methylation during Src-induced transformation.

Src, a nonreceptor tyrosine kinase, was the first oncogene identified from an oncogenic virus. Mechanistic studies of Src-induced transformations aid in understanding the pathologic processes underlying tumorigenesis and may provide new strategies for cancer therapy. Although several pathways and protein modifications are reportedly involved in Src-induced transformation, the detailed mechanisms of their regulation remain unclear. Protein methylation is an important PTM that is widely involved in cellular physiology. In this study, we determined if protein methylation was involved in Src activation and which methylated proteins were associated with this activity. Using in vitro methylation and 2-DE analysis of viral Src (v-Src)-transformed rat kidney epithelial cells (RK3E), several known and novel methylated proteins were identified based on their changes in methylation signal intensity upon transformation. Among these, elongation factor 2 (EF-2), heterogeneous nuclear ribonucleoprotein K (hnRNP K), and ß-tubulin protein expressions remained unchanged, indicating that their altered methylation levels were due to Src activation. In addition, the altered expression of ß-actin, vimentin, and protein phosphatase 2, catalytic subunit (PPP2C) as well as protein phosphatase 2, catalytic subunit methylation were also confirmed in RK3E cells transformed with a human oncogenic Src mutant (Src531), supporting their association with Src-induced transformation in human cancer. Together, we showed putative involvement of protein methylation in Src activation and our identification of methylated proteins provides important targets for extensively studying Src-induced transformations.

Identification of the methylation preference region in heterogeneous nuclear ribonucleoprotein K by protein arginine methyltransferase 1 and its implication in regulating nuclear/cytoplasmic distribution.

Protein arginine methylation plays crucial roles in numerous cellular processes. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein participating in a variety of cellular functions including transcription and RNA processing. HnRNP K is methylated at multiple sites in the glycine- and arginine-rich (RGG) motif. Using various RGG domain deletion mutants of hnRNP K as substrates, here we show by direct methylation assay that protein arginine methyltransferase 1 (PRMT1) methylated preferentially in a.a. 280-307 of the RGG motif. Kinetic analysis revealed that deletion of a.a. 280-307, but not a.a. 308-327, significantly inhibited rate of methylation. Importantly, nuclear localization of hnRNP K was significantly impaired in mutant hnRNP K lacking the PRMT1 methylation region or upon pharmacological inhibition of methylation. Together our results identify preferred PRMT1 methylation sequences of hnRNP K by direct methylation assay and implicate a role of arginine methylation in regulating intracellular distribution of hnRNP K.

Establishment of an ectopically expressed and functional PRMT1 for proteomic analysis of arginine-methylated proteins.

Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), plays crucial roles in a variety of cellular processes. Mammalian PRMT1 exists in a large protein complex in cells, which has been implied in modulating the regulatory and catalytic properties of this enzyme. Establishment of a mammalian comparative approach will help to identify putative substrates of PRMT1 in an authentic condition. Here, we showed that ectopically expressed PRMT1 in mammalian HEK293 cells not only exhibited catalytic properties comparable to the endogenous enzyme but also existed in a functional complex together with endogenous PRMT1 and thus functioned as an endogenous counterpart. In addition, the measured methylation level of cellular proteins using a tritium-labeled methyl donor was accordingly enhanced upon ectopic expression of PRMT1. Subsequent proteomic analysis with such PRMT1-expressing cells allowed us to identify several known and putative methylated proteins. In vitro methylation of selected proteins, eukaryotic translation initiation factor 4A-I and vimentin, by cellular PRMT1 was shown. Together, we have demonstrated the functional equivalence of ectopically expressed PRMT1 in HEK293 cells and its application to systematically identify the substrate proteins in a mammalian cell context.

Protein-arginine methyltransferase 1 suppresses megakaryocytic differentiation via modulation of the p38 MAPK pathway in K562 cells.

Protein-arginine methyltransferase 1 (PRMT1) plays pivotal roles in various cellular processes. However, its role in megakaryocytic differentiation has yet to be investigated. Human leukemia K562 cells have been used as a model to study hematopoietic differentiation. In this study, we report that ectopic expression of HA-PRMT1 in K562 cells suppressed phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation as demonstrated by changes in cytological characteristics, adhesive properties, and CD41 expression, whereas knockdown of PRMT1 by small interference RNA promoted differentiation. Impairment of the methyltransferase activity of PRMT1 diminished the suppressive effect. These results provide evidence for a novel role of PRMT1 in negative regulation of megakaryocytic differentiation. Activation of ERK MAPK has been shown to be essential for megakaryocytic differentiation, although the role of p38 MAPK is still poorly understood. We show that knockdown of p38alpha MAPK or treatment with the p38 inhibitor SB203580 significantly enhanced PMA-induced megakaryocytic differentiation. Further investigation revealed that PRMT1 promotes activation of p38 MAPK without inhibiting activation of ERK MAPK. In p38alpha knockdown cells, PRMT1 could no longer suppress differentiation. In contrast, enforced expression of p38alpha MAPK suppressed PMA-induced megakaryocytic differentiation of parental K562 as well as PRMT1-knockdown cells. We propose modulation of the p38 MAPK pathway by PRMT1 as a novel mechanism regulating megakaryocytic differentiation. This study thus provides a new perspective on the promotion of megakaryopoiesis.

Mitochondrial anchoring of PKCalpha by PICK1 confers resistance to etoposide-induced apoptosis.

Various pathways, including regulation of functions of the Bcl-2 family, are implicated in the survival promotion by PKCalpha, however the molecular mechanisms are still obscure. We have previously demonstrated that PKCalpha is selectively anchored to mitochondria by PICK1 in fibroblasts NIH 3T3. In this study, we show that over-expression of PICK1 in leukemia REH confers resistance to etoposide-induced apoptosis, which requires an interaction with PKCalpha as the non-interacting mutant PICK1 loses the pro-survival activity. The PKCalpha selective inhibitor Gö6976 also abolishes the anti-apoptotic effect indicating a requirement for PKC activity. Disruption of PICK1/PKCalpha interactions by inhibitory peptides significantly increases cellular susceptibility to etoposide. Similar effects are also observed in HL60 cells, which exhibit an intrinsic resistance to etoposide. Molecular analysis shows that the wild type PICK1, but not the non-interacting mutant, prevents the loss of mitochondrial membrane potential with a coincident increase in phosphorylation of the anti-apoptotic Bcl-2(Ser70) and a decrease in dimerization of the pro-apoptotic Bax. PICK1 may provide the spatial proximity for phosphorylation of Bcl-2(Ser70) by PKCalpha which then leads to a higher survival. Taken together, our results suggest that PICK1 may mediate the pro-survival activity of PKCalpha by serving as a molecular link between PKCalpha and mitochondria.

Direct mass-spectrometric identification of Arg296 and Arg299 as the methylation sites of hnRNP K protein for methyltransferase PRMT1.

Protein methylation is one of the most important post-translational modifications that contribute to the diversity and complexity of proteome. Here we report the study of in vitro methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) with protein arginine methyltransferase 1 (PRMT1), as an enzyme, and S-adenosyl-L-methionine (SAM), as a methyl donor. The mass analysis of tryptic peptides of hnRNP K before and after methylation reveals the addition of four methyl groups in the residues 288-303. Tandem mass-spectrometric analysis of this peptide shows that both Arg296 and Arg299 are dimethylated. In addition, fragmentation analysis of such methylated arginines illustrate that they are both asymmetric dimethylarginines. Since Arg296 and Arg299 are located near the SH3-binding domains of hnRNP K, such methylation has the potential in regulating the interaction of hnRNP K with Src protein family. Our results provide crucial information for further functional study of hnRNP K methylation.

Identification of V23RalA-Ser194 as a critical mediator for Aurora-A-induced cellular motility and transformation by small pool expression screening.

Human Aurora kinases have three gene family members: Aurora-A, Aurora-B, and Aurora-C. It is not yet established what the specificity of these kinases are and what signals relayed by their reactions. Therefore, we employed small pool expression screening to search for downstream substrates of Aurora-A. Interestingly, all of the identified Aurora-A substrates were resistant to serve as substrates for Aurora-B or Aurora-C, suggesting that these Aurora family members may have distinct substrate specificity for propagation of diverse signaling pathways, even though they share a conserved catalytic kinase domain. Of the candidate substrates, Aurora-A could increase the functional activity of RalA. Mutational analysis revealed that RalA-Ser194 was the phosphorylation site for Aurora-A. Ectopic expression of V23RalA-WT could enhance collagen I-induced cell migration and anchorage-independent growth in Madin-Darby canine kidney (MDCK) Aurora-A stable cell lines. In contrast, overexpression of V23RalA-S194A in MDCK Aurora-A-stable cell lines abolished the intrinsic migration and transformation abilities of Aurora-A. To our knowledge, this is the first systematic search for the downstream substrates of Aurora-A kinase. Moreover, these results support the notion that Aurora-A may act in concert with V23RalA through protein phosphorylation on Ser194 to promote collagen I-induced cell motility and anchorage-independent growth in MDCK epithelial cells.

Identification of the substrates and interaction proteins of aurora kinases from a protein-protein interaction model.

The increasing use of high-throughput and large-scale bioinformatics-based studies has generated a massive amount of data stored in a number of different databases. The major need now is to explore this disparate data to find biologically relevant interactions and pathways. Thus, in the post-genomic era, there is clearly a need for the development of algorithms that can accurately predict novel protein-protein interaction networks in silico. The evolutionarily conserved Aurora family kinases have been chosen as a model for the development of a method to identify novel biological networks by a comparison of human and various model organisms. Our search methodology was designed to predict and prioritize molecular targets for Aurora family kinases, so that only the most promising are subjected to empirical testing. Four potential Aurora substrates and/or interacting proteins, TACC3, survivin, Hec1, and hsNuf2, were identified and empirically validated. Together, these results justify the timely implementation of in silico biology in routine wet-lab studies and have also allowed the application of a new approach to the elucidation of protein function in the post-genomic era.

PICK1, an anchoring protein that specifically targets protein kinase Calpha to mitochondria selectively upon serum stimulation in NIH 3T3 cells.

PICK1 binds to protein kinase Calpha (PKCalpha) through the carboxylate-binding loop in its PDZ (PSD95/Disc-large/ZO-1) domain and the C terminus of PKCalpha. We have previously shown that PICK1 modulates the catalytic activity of PKC selectively toward the antiproliferative gene TIS21. To investigate whether PICK1 plays a role in targeting activated PKCalpha to a particular intracellular compartment in addition to regulating PKC activity, we examine the localization of PICK1 and PKCalpha in response to various stimuli. Double staining with organelle markers and anti-rPICK1 antibodies reveals that PICK1 is associated with mitochondria but not with endoplasmic reticulum or Golgi in NIH 3T3 cells. Deletion of the PDZ domain impairs the mitochondria localization of PICK1, whereas mutations in the carboxylate-binding loop do not have an effect, suggesting that PICK1 can bind PKCalpha and mitochondria simultaneously. Upon serum stimulation, PICK1 translocates and displays a dense ring-like structure around the nucleus, where it still associates with mitochondria. A substantial portion of PKCalpha is concomitantly found in the condense perinuclear region. The C terminal-deleted PKCalpha fails to translocate and remains a diffuse cytoplasmic distribution, indicating that a direct interaction between PICK1 and PKCalpha is required for PKCalpha anchoring to mitochondria. 12-O-Tetradecanoylphorbol-13-acetate stimulation, in contrast, causes translocation of PKCalpha to the plasma membrane, whereas the majority of PICK1 remains in a cytoplasmic punctate pattern. Deletion at the C terminus of PKCalpha has no effect on 12-O-tetradecanoylphorbol-13-acetate-induced translocation. These findings indicate a previously unidentified role for PICK1 in anchoring PKCalpha to mitochondria in a ligand-specific manner.

Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

BACKGROUND: Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals. RESULTS: The linker protein, a monoclonal antibody (mAb C), is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57) of transgenic pigs (F0 generation). CONCLUSIONS: Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.