Hematech Inc.

Sioux Falls, SD, United States

Hematech Inc.

Sioux Falls, SD, United States
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Kazuki Y.,Institute of Regenerative Medicine and Biofunction | Kazuki Y.,Tottori University | Kobayashi K.,Chiba University | Aueviriyavit S.,Chiba University | And 19 more authors.
Human Molecular Genetics | Year: 2013

Human CYP3A is the most abundant P450 isozyme present in the human liver and small intestine, and metabolizes around 50% of medical drugs on the market. The human CYP3A subfamily comprises four members (CYP3A4, CYP3A5, CYP3A7, CYP3A43) encoded on human chromosome 7. However, transgenic mouse lines carrying the entire human CYP3A cluster have not been constructed because of limitations in conventional cloning techniques. Here, we show that the introduction of a human artificial chromosome (HAC) containing the entire genomic human CYP3A locus recapitulates tissue- and stage-specific expression of human CYP3A genes and xenobiotic metabolism in mice. About 700 kb of the entire CYP3A genomic segment was cloned into a HAC (CYP3A-HAC), and trans-chromosomic (Tc) mice carrying a single copy of germline-transmittable CYP3A-HAC were generated via a chromosome-engineering technique. The tissue- and stage-specific expression profiles of CYP3A genes were consistent with those seen in humans. We further generated mice carrying the CYP3A-HAC in the background homozygous for targeted deletion of most endogenous Cyp3a genes. In this mouse strain with 'fully humanized' CYP3A genes, the kinetics of triazolam metabolism, CYP3A-mediated mechanism-based inactivation effects and formation of fetal-specific metabolites of dehydroepiandrosterone observed in humans were well reproduced. Thus, these mice are likely to be valuable in evaluating novel drugs metabolized by CYP3A enzymes and in studying the regulation of human CYP3A gene expression. Furthermore, this system can also be used for generating Tc mice carrying other human metabolic genes. © The Author 2012. Published by Oxford University Press. All rights reserved.

Matsushita H.,Sanford Applied Biosciences L.L.C. | Matsushita H.,Hematech Inc. | Sano A.,Chiyoda Corporation | Sano A.,Hematech Inc. | And 11 more authors.
PLoS ONE | Year: 2014

Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM-/-, bIGHML1-/-; double knockouts or DKO). However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the hIGK and hIGL genomic loci on the HAC and from the endogenous bovine kappa-chain (bIGK) and lambda-chain (bIGL) genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ) and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ). To improve fully hIgG production in Tc cattle, we here report the deletion of the entire bIGL joining (J) and constant (C) gene cluster (b IGLJ1-IGLC1 to bIGLJ5-IGLC5) by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (bIGHM-/-, bIGHML1-/- and bIGL-/-; TKO) Tc cattle. We further demonstrate that bIGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ). © 2014 Matsushita et al.

Sano A.,Chiyoda Corporation | Sano A.,Hematech Inc. | Matsushita H.,Sanford Applied Biosciences L.L.C. | Matsushita H.,Hematech Inc. | And 12 more authors.
PLoS ONE | Year: 2013

Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin heavy-chain (hIGH) and kappa-chain (hIGK) germline loci (named as κHAC) are capable of producing functional hpAbs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, are homozygously inactivated (double knockouts or DKO). However, B lymphocyte development in these Tc cattle is compromised, and the overall production of hpAbs is low. Here, we report the construction of an improved HAC, designated as cKSL-HACΔ, by incorporating all of the human immunoglobulin germline loci into the HAC. Furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (hIgM) and bovine transmembrane α and β immunoglobulins (bIgα and bIgβ) in the pre-B cell receptor (pre-BCR) complex, we partially replaced (bovinized) the hIgM constant domain with the counterpart of bovine IgM (bIgM) that is involved in the interaction between bIgM and bIgα/Igβ; human IgM bovinization would also improve the functionality of hIgM in supporting B cell activation and proliferation. We also report the successful production of DKO Tc cattle carrying the cKSL-HACΔ (cKSL-HACΔ/DKO), the dramatic improvement of B cell development in these cattle and the high level production of hpAbs (as measured for the human IgG isotype) in the plasma. We further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human IgG (hIgG) can be produced from such Tc cattle. © 2013 Sano et al.

Rodriguez-Sosa J.R.,University of Pennsylvania | Rodriguez-Sosa J.R.,University of Calgary | Rathi R.,University of Pennsylvania | Wang Z.,Hematech Inc | And 2 more authors.
Journal of Andrology | Year: 2011

Testis tissue xenografting represents a versatile model to study testis biology, and to preserve fertility in immature animals. To evaluate whether bovine fetal testes can mature when grafted into mouse hosts, small fragments of testes from midgestation (125 to 145 days of gestation) bovine fetuses were grafted ectopically into immunodeficient castrated male mice. At grafting, donor tissue displayed the typical seminiferous cords composed of gonocytes and primitive Sertoli cells. At 5 or 10 months after grafting, weight of the seminal vesicles in recipient mice was indicative of production of bioactive testosterone by xenografts. Xenografts showed similar development regardless of donor age. At 5 months, tubule formation occurred but germ cell differentiation had not proceeded beyond the spermatogonia stage. At 10 months, an increase in tubule size was evident and pachytene spermatocytes were observed as the most advanced type of germ cells in the xenografts of 2 donors. The number of tubules with germ cells was reduced in xenografts compared to donor tissue, but at 10months the number of germ cells per tubule was higher than in donors. Germ cell proliferation was similar in donor tissue and xenografts. However, Sertoli cells showed a higher proliferation rate in xenografts collected at 5 months than in donor fetal testes and xenografts collected at 10 months. Sertoli cells in xenografts showed a progressive but incomplete loss of expression of Müllerian inhibiting substance and weak androgen receptor expression, indicating an incomplete Sertoli cell maturation. In conclusion, fetal testis tissue developed partially, qualitatively similar to pubertal testes in situ. © American Society of Andrology.

McLean C.A.,Agresearch Ltd. | Wang Z.,Hematech Inc. | Babu K.,Agresearch Ltd. | Edwards A.,Hematech Inc. | And 3 more authors.
Animal Reproduction Science | Year: 2010

If the full potential of chromatin transfer (CT) technology is to be realized for both animal production and biomedical applications it is imperative that the efficiency of the reprogramming process be improved, and the potential for deleterious development be eliminated. Generation of the first cloned animals from adult somatic cells demonstrated that development is substantially an epigenetic process (Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH, 1997. Viable offspring derived from fetal and adult mammalian cells. Nature. 385(6619): 810-813.). In this study, we provide preliminary evidence that the epigenetic state of the donor cell, may be valuable in assessing potential cloning success. We have measured key indicators of cellular epigenetic state in both serially derived cell populations of the same genetic origin, but differing in epigenomic status, and in a distinct cohort of donor cell populations with diverse genetic origins and epigenomic status. Specifically, the relative abundance of particular histone modifications in donor populations prior to manipulation has been correlated with the measurable variance in reprogramming efficiencies observed following CT, as defined by the number of resulting live births and healthy progeny, and the concomitant incidence of deleterious growth measures (notably the appearance of large offspring syndrome (LOS)). Thus, we suggest that the likely outcome and relative success of cloning may be predictable based on the expression of discriminating histone marks present in the donor cell population before CT. This approach may provide the basis of a prognostic signature for the future evaluation and risk assessment of putative donor cells prior to CT, and thus increase future cloning success and alleviate the incidence of abnormal development. © 2009 Elsevier B.V. All rights reserved.

Liu G.E.,U.S. Department of Agriculture | Hou Y.,U.S. Department of Agriculture | Hou Y.,University of Maryland University College | Robl J.M.,Hematech Inc | And 2 more authors.
Genome Integrity | Year: 2011

Background: Transgenic cattle carrying multiple genomic modifications have been produced by serial rounds of somatic cell chromatin transfer (cloning) of sequentially genetically targeted somatic cells. However, cloning efficiency tends to decline with the increase of rounds of cloning. It is possible that multiple rounds of cloning compromise the genome integrity or/and introduce epigenetic errors in the resulting cell lines, rendering a decline in cloning. To test these possibilities, we performed 9 high density array Comparative Genomic Hybridization (CGH) experiments to test the genome integrity in 3 independent bovine transgenic cell lineages generated from genetic modification and cloning. Our plan included the control hybridizations (self to self) of the 3 founder cell lines and 6 comparative hybridizations between these founders and their derived cell lines with either high or low cloning efficiencies.Results: We detected similar amounts of differences between the control hybridizations (8, 13 and 39 differences) and the comparative analyses of both "high" and "low" cell lines (ranging from 7 to 57 with a mean of ~20). Almost 75% of the large differences (>10 kb) and about 45% of all differences shared the same type (loss or gain) and were located in nearby genomic regions across hybridizations. Therefore, it is likely that they were not true differences but caused by systematic factors associated with local genomic features (e.g. GC contents).Conclusions: Our findings reveal that large copy number variations are less likely to arise during genetic targeting and serial rounds of cloning, fortifying the notion that epigenetic errors introduced from serial cloning may be responsible for the cloning efficiency decline. © 2011 Liu et al; licensee BioMed Central Ltd.

Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described. © 2011 Springer Science+Business Media, LLC.

This chapter describes a detailed protocol for deriving mouse embryonic stem (ES) cell lines from blastocysts that are produced either by fertilization or by somatic cell nuclear transfer (NT or cloning). Rather than function as a scientific communication with the experts in this field, this protocol is written with researchers who are new to mouse ES cell line derivation in mind. Therefore, researchers who want to establish such methods in laboratories where mouse ES cell line derivation has never been done before should find this chapter helpful. © 2011 Springer Science+Business Media, LLC.

Wang J.,University of Wisconsin - Madison | Liu Y.,University of Wisconsin - Madison | Li Z.,University of Wisconsin - Madison | Wang Z.,Hematech Inc. | And 8 more authors.
Blood | Year: 2011

Both monoallelic and biallelic oncogenic NRAS mutations are identified in human leukemias, suggesting a dose-dependent role of oncogenic NRAS in leukemogenesis. Here, we use a hypomorphic oncogenic Nras allele and a normal oncogenic Nras allele (Nras G12Dhypo and Nras G12D, respectively) to create a gene dose gradient ranging from 25% to 200% of endogenous Nras G12D/+. Mice expressing Nras G12Dhypo/G12Dhypo develop normally and are tumor-free, whereas early embryonic expression of Nras G12D/+ is lethal. Somatic expression of Nras G12D/G12D but not Nras G12D/+ leads to hyperactivation of ERK, excessive proliferation of myeloid progenitors, and consequently an acute myeloproliferative disease. Using a bone marrow transplant model, we previously showed that ∼ 95% of animals receiving Nras G12D/+ bone marrow cells develop chronic myelomonocytic leukemia (CMML), while ∼ 8% of recipients develop acute T-cell lymphoblastic leukemia/lymphoma [TALL] (TALL-het). Here we demonstrate that 100% of recipients transplanted with Nras G12D/G12D bone marrow cells develop TALL (TALL-homo). Although both TALL-het and -homo tumors acquire Notch1 mutations and are sensitive to a γ-secretase inhibitor, endogenous Nras G12D/+ signaling promotes TALL through distinct genetic mechanism(s) from Nras G12D/G12D. Our data indicate that the tumor transformation potential of endogenous oncogenic Nras is both dose- and cell type-dependent. © 2011 by The American Society of Hematology.

PubMed | Hematech Inc.
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2011

This chapter describes a detailed protocol for deriving mouse embryonic stem (ES) cell lines from blastocysts that are produced either by fertilization or by somatic cell nuclear transfer (NT or cloning). Rather than function as a scientific communication with the experts in this field, this protocol is written with researchers who are new to mouse ES cell line derivation in mind. Therefore, researchers who want to establish such methods in laboratories where mouse ES cell line derivation has never been done before should find this chapter helpful.

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