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Jose Cadalso’s Cartas marruecas (1789) and Benito Perez Galdos’s Aita Tettauen (1905) share a common figure: the ostensibly fake Moor. Through the analytical trope of burlesque performance, I show how the production of Moorish “Otherness” is inextricably entangled with the articulation of Spanish identity in the novels. The epistolary performances of the African Moorish characters alternately spotlight and disguise the making of difference in a process I refer to as “cultural burlesque.” These narrative performances tease the reader with glimpses of purported “authenticity,” cultivating identitary ambiguity in a manner that interrogates and unsettles binary paradigms presumed to be natural or essential (Spanish/Moroccan, European/African, Catholic/Muslim). By calling attention to the performativity of national, cultural, and religious identities, the novels convey critiques of the Spanish societies of both the 18th and 20th centuries; as scholars like Susan Martin-Marquez have noted, politicians and intellectuals in both moments responded to issues of national identity and European modernity through a paradoxical embrace and denial of the Moorish figure—and by extension Spain’s African heritage. The presence of these performances of cultural burlesque in two works separated by more than a century points to a transhistorical Spanish discursive tendency to domesticate and/or colonize the Maghribian Moor in the service of soothing internal and international ills.

Human embryonic stem cells and induced pluripotent stem cells (hESCs and hiPSCs) are powerful tools for biomedical research. Realizing the full potential of these cells requires efficient genetic modification. However, techniques to generate cell type specific lineage reporters as well as reliable tools to disrupt, repair or overexpress genes by gene targeting are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc finger nuclease (ZFN) mediated genome editing. First, using ZFNs specific for the OCT4 locus we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Secondly, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.

Rett Syndrome (RTT) is a severe form of X-linked mental retardation caused by mutations in the gene coding for methyl CpG-binding protein 2 (MECP2). Mice deficient in MeCP2 have a range of physiological and neurological abnormalities that mimic the human syndrome. Here we show that systemic treatment of MeCP2 mutant mice with an active peptide fragment of Insulin-like Growth Factor 1 (IGF-1) extends the life span of the mice, improves locomotor function, ameliorates breathing patterns, and reduces irregularity in heart rate. In addition, treatment with IGF-1 peptide increases brain weight of the mutant mice. Multiple measurements support the hypothesis that RTT results from a deficit in synaptic maturation in the brain: MeCP2 mutant mice have sparse dendritic spines and reduced PSD-95 in motor cortex pyramidal neurons, reduced synaptic amplitude in the same neurons, and protracted cortical plasticity in vivo. Treatment with IGF-1 peptide partially restores spine density and synaptic amplitude, increases PSD-95, and stabilizes cortical plasticity to wild-type levels. Our results thus strongly suggest IGF-1 as a candidate for pharmacological treatment of RTT and potentially of other CNS disorders caused by delayed synapse maturation.

We develop biodegradable polymeric nanoparticles to facilitate nonviral gene transfer to human embryonic stem cells (hESCs). Small (approximately 200 nm), positively charged (approximately 10 mV) particles are formed by the self assembly of cationic, hydrolytically degradable poly(beta-amino esters) and plasmid DNA. By varying the end group of the polymer, we can tune the biophysical properties of the resulting nanoparticles and their gene-delivery efficacy. We created an OCT4-driven GFP hES cell line to allow the rapid identification of nanoparticles that facilitate gene transfer while maintaining an hESC undifferentiated state. Using this cell system, we synthesized nanoparticles that have gene delivery efficacy that is up to 4 times higher than that of the leading commercially available transfection agent, Lipofectamine 2000. Importantly, these materials have minimal toxicity and do not adversely affect hESC colony morphology or cause nonspecific differentiation.

In humans, mutations in the X-linked MECP2 gene, are the cause of Rett syndrome (RTT), a neurodevelopmental disorder that affects mainly girls. MeCP2 binds to methylated CpGs and is thought to act as a transcriptional repressor. In male mice, deletion or targeted mutation of Mecp2 leads to lethality and causes a neuronal phenotype. Selective mutation of Mecp2 in postnatal neurons results in a similar, although delayed, phenotype, suggesting that the symptoms are caused by MeCP2 deficiency in postmitotic neurons. In agreement with this idea, expression of a Mecp2 transgene in postmitotic neurons of Mecp2 -null mutant mice resulted in the phenotypical rescue of the symptoms. To assess whether postnatal activation of MeCP2 in mutant animals could also affect the progression of the disorder, we constructed a conditionally active Mecp2 “rescue transgene” that was activated between P0 and P30. The Mecp2 transgene was under the control of the CAGGS promoter and was activated by using brain specific Cre-mediated recombination. Our results indicate that postnatal, neuron-specific activation of MeCP2 as late as 2–4 weeks of age significantly prolonged the lifespan of mutant animals and delayed the onset of neurologic symptoms.

Transcriptional silencing mediated by DNA methylation is a critical component of epigenetic regulation during early embryonic development in animals. However, the requirement for DNA methylation during activation and differentiation of mature CD8+ T cells into effector and memory cells is not clear. Using cre-mediated deletion of DNA methyltransferase 1 (Dnmt1) at the time of CD8+ T cell activation, we investigated the obligation for maintaining patterns of DNA methylation during the generation of Ag-specific effector and memory CD8+ T cells in response to acute viral infection of mice with lymphocytic choriomeningitis virus. Dnmt1−/− CD8+ T cells failed to undergo the massive CD8+ T cell expansion characteristic of lymphocytic choriomeningitis virus infection, leading to >80% reductions in Ag-specific effector CD8+ T cells at the height of the response. Despite this, Dnmt1−/− CD8+ T cells efficiently controlled the viral infection. Interestingly, the number of Ag-specific Dnmt1−/− memory CD8+ T cells was moderately reduced compared with the reductions seen at day 8 postinfection. Our data suggest that ablation of Dnmt1 and subsequent DNA methylation affect the finite proliferative potential of Ag-specific CD8+ T cells with moderate effects on their differentiation to effector and memory CD8+ T cells.

Summary The POU-domain transcription factor Oct-4 is normally expressed in pluripotent cells of the mammalian embryo. In addition, germ-cell tumors and a few somatic tumors show detectable expression of Oct-4 . While Oct-4's role during preimplantation development is to maintain embryonic cells in a pluripotent state, little is known about its potential oncogenic properties. Here we investigate the effect of ectopic Oct-4 expression on somatic tissues of adult mice using a doxycycline-dependent expression system. Activation of Oct-4 results in dysplastic growths in epithelial tissues that are dependent on continuous Oct-4 expression. Dysplastic lesions show an expansion of progenitor cells and increased β-catenin transcriptional activity. In the intestine, Oct-4 expression causes dysplasia by inhibiting cellular differentiation in a manner similar to that in embryonic cells. These data show that certain adult progenitors remain competent to interpret key embryonic signals and support the notion that progenitor cells are a driving force in tumorigenesis.

Regulated apoptosis is essential for both the development and the subsequent maintenance of the immune system. Interleukins, including IL-2, IL-4, IL-7 and IL-15, heavily influence lymphocyte survival during the vulnerable stages of VDJ rearrangement and later in ensuring cellular homeostasis, but the genes specifically responsible for the development and maintenance of lymphocytes have not been identified. The antiapoptotic protein MCL-1 is an attractive candidate, as it is highly regulated, appears to enhance short-term survival and functions at an apical step in genotoxic deaths. However, Mcl-1 deficiency results in peri-implantation lethality. Here we show that mice conditional for Mcl-1 display a profound reduction in B and T lymphocytes when MCL-1 is removed. Deletion of Mcl-1 during early lymphocyte differentiation increased apoptosis and arrested the development at pro-B-cell and double-negative T-cell stages. Induced deletion of Mcl-1 in peripheral B- and T-cell populations resulted in their rapid loss. Moreover, IL-7 both induced and required MCL-1 to mediate lymphocyte survival. Thus, MCL-1, which selectively inhibits the proapoptotic protein BIM, is essential both early in lymphoid development and later on in the maintenance of mature lymphocytes.

Proviruses carrying drug-inducible Oct4, Sox2, Klf4 and c-Myc used to derive 'primary' induced pluripotent stem (iPS) cells were segregated through germline transmission, generating mice and cells carrying subsets of the reprogramming factors. Drug treatment produced 'secondary' iPS cells only when the missing factor was introduced. This approach creates a defined system for studying reprogramming mechanisms and allows screening of genetically homogeneous cells for compounds that can replace any transcription factor required for iPS cell derivation.

Molecular complementation of mutant phenotypes by transgenic technology is a potentially important tool for gene identification. A technology was developed that allows the transfer of a physically intact yeast artificial chromosome (YAC) into the germ line of the mouse. A purified 150-kilobase YAC encompassing the murine gene Col1a1 was efficiently introduced into embryonic stem (ES) cells via lipofection. Chimeric founder mice were derived from two transfected ES cell clones. These chimeras transmitted the full length transgene through the germ line, generating two transgenic mouse strains. Transgene expression was visualized as nascent transcripts in interphase nuclei and quantitated by ribonuclease protection analysis. Both assays indicated that the transgene was expressed at levels comparable to the endogenous collagen gene.

We report a novel transgenic mouse model for direct reprogramming of somatic cells by expressing four reprogramming factors from a single genomic locus using a drug-inducible transgene. Multiple somatic cell types explanted from different tissues can generate induced pluripotent stem cells (iPSC) by culture in doxycycline (Dox). Because the reprogramming factors are carried on a single polycistronic construct the transgenic mice can be easily maintained and transferred into another background.

Two parvovirus 1319 cDNA libraries have been constructed; one from COS-7 cells transfected with a B19/pSVOd hybrid vector and the other from B19-infected human erythroid leukemic cells. We have used these libraries to investigate the expression of the abundant classes of polyadenylated 1319 RNAs; the 700- and 800-nt class which terminates in the middle of the genome and the 500- and 600-nt class which contains an ORF from the extreme right-hand end of the genome. The 700- and 800-nt RNA species were not found in the COS cell library, suggesting that a variant polyadenylation signal (ATTAAA or AATAAC) in the middle of the genome is not efficiently recognized in these cells. In contrast, the 700- and 800-nt class was highly represented in the human library, confirming the use of this variant polyadenylation signal in the normal host cell of the virus. In COS cells the middle exon of the 500- and 600-nt class of RNA exhibited variability in both splice donor and acceptor sites. However, in human cells there were only two splice acceptor sites nt 1910 and 2030, and a single splice donor site nt 2183 for this exon. Antisera, prepared against a peptide derived from the 94-aa potential protein encoded by the 500- and 600-nt class of RNA, recognized, on a Western blot, a polypeptide of approximately 11 kDa that was translated in vitro from these cDNAs and in vivo in pSVOd/B19 transfected COS cells. Immunoprecipitation revealed that two proteins were made from this ORF, suggesting the use of internal translation initiation site(s). Another antisera, raised against a second peptide corresponding to an antigenic region of the potential protein encoded by the 700- and 800-nt class of RNA, failed to detect a 15-kDa protein by Western blotting or immunoprecipitation of labeled proteins both in vitro and in vivo in COS cells.

SummaryInduced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.

It has recently been demonstrated that mouse and human fibroblasts can be reprogrammed into an embryonic stem cell–like state by introducing combinations of four transcription factors. However, the therapeutic potential of such induced pluripotent stem (iPS) cells remained undefined. By using a humanized sickle cell anemia mouse model, we show that mice can be rescued after transplantation with hematopoietic progenitors obtained in vitro from autologous iPS cells. This was achieved after correction of the human sickle hemoglobin allele by gene-specific targeting. Our results provide proof of principle for using transcription factor–induced reprogramming combined with gene and cell therapy for disease treatment in mice. The problems associated with using retroviruses and oncogenes for reprogramming need to be resolved before iPS cells can be considered for human therapy.

X chromosome inactivation in mammals requires expression of the gene Xist, which maps to the X chromosome inactivation centre (Xic) and encodes an untranslated RNA. Truncation of Xist RNA by gene targeting is lethal for female embryos and prevents the inactivation of the X chromosome carrying the deletion. This indicates that Xist RNA is necessary for initiation and propagation of the inactivation process. Xist is transcribed from the inactive X and its expression is silenced by DNA methylation, suggesting that methylation is crucial for shielding the active X chromosome against the inactivation process. Gene transfer experiments using transgenes the size of yeast artificial chromosomes have determined that a 450 kb fragment of DNA carrying Xist acts as an inactivation centre and is sufficient for initiation, propagation and maintenance of the inactive state. The elements for counting and choosing X chromosomes are part of the transgene. We have shown that X inactivation is mediated by a post-translational mechanism, i.e. the stabilization of Xist RNA, rather than by the regulation of the Xist promoter.

Transgenic and gene-targeted mutant mice provide powerful tools for analysis of the cellular processes involved in early development and in the pathogenesis of many diseases. Here we describe a transgene integration strategy mediated by site-specific recombination that allows establishment of multiple embryonic stem (ES) cell lines carrying tetracycline-inducible genes targeted to a specific locus to assure predictable temporal and spatial expression in ES cells and mice. Using homologous recombination we inserted an frt homing site into which tetracycline-inducible transgenes can be integrated efficiently in the presence of FLPe recombinase. This strategy and the vectors described here are generally applicable to any locus in ES cells and should allow for the rapid production of mice with transgenes efficiently targeted to a defined site.

Mutations in MECP2 are the cause of Rett syndrome (RTT) in humans, a neurodevelopmental disorder that affects mainly girls. MeCP2 is a protein that binds CpG dinucleotides and is thought to act as a global transcriptional repressor. It is highly expressed in neurons, but not in glia, of the postnatal brain. The timing of MeCP2 activation correlates with the maturation of the central nervous system, and recent reports suggest that MeCP2 may be involved in the formation of synaptic contacts and may function in activity-dependent neuronal gene expression. Deletion or targeted mutation of Mecp2 in mice leads to a Rett-like phenotype. Selective mutation of Mecp2 in postnatal neurons leads to a similar, although delayed, phenotype, suggesting that MeCP2 plays a role in postmitotic neurons. Here we test the hypothesis that the symptoms of RTT are exclusively caused by a neuronal MeCP2 deficiency by placing Mecp2 expression under the control of a neuron-specific promoter. Expression of the Mecp2 transgene in postmitotic neurons resulted in symptoms of severe motor dysfunction. Transgene expression in Mecp2 mutant mice, however, rescued the RTT phenotype.

DNA methyltransferase I (Dnmt1), the maintenance enzyme for DNA cytosine methylation, is expressed at high levels in the CNS during embryogenesis and after birth. Because embryos deficient for Dnmt1 die at gastrulation, the role of Dnmt1 in the development and function of the nervous system could not be studied by using this mutation. We therefore used the cre/loxP system to produce conditional mutants that lack Dnmt1 in neuroblasts of embryonic day 12 embryos or in postmitotic neurons of the postnatal animal. Conditional deletion of the Dnmt1 gene resulted in rapid depletion of Dnmt1 proteins, indicating that the enzyme in postmitotic neurons turns over quickly. Dnmt1 deficiency in postmitotic neurons neither affected levels of global DNA methylation nor influenced cell survival during postnatal life. In contrast, Dnmt1 deficiency in mitotic CNS precursor cells resulted in DNA hypomethylation in daughter cells. Whereas mutant embryos carrying 95% hypomethylated cells in the brain died immediately after birth because of respiratory distress, mosaic animals with 30% hypomethylated CNS cells were viable into adulthood. However, these mutant cells were eliminated quickly from the brain within 3 weeks of postnatal life. Thus, hypomethylated CNS neurons were impaired functionally and were selected against at postnatal stages.

Cytosine methylation of mammalian DNA is essential for the proper epigenetic regulation of gene expression and maintenance of genomic integrity. To define the mechanism through which demethylated cells die, and to establish a paradigm for identifying genes regulated by DNA methylation, we have generated mice with a conditional allele for the maintenance DNA methyltransferase gene Dnmt1. Cre-mediated deletion of Dnmt1 causes demethylation of cultured fibroblasts and a uniform p53-dependent cell death. Mutational inactivation of Trp53 partially rescues the demethylated fibroblasts for up to five population doublings in culture. Oligonucleotide microarray analysis showed that up to 10% of genes are aberrantly expressed in demethylated fibroblasts. Our results demonstrate that loss of Dnmt1 causes cell-type-specific changes in gene expression that impinge on several pathways, including expression of imprinted genes, cell-cycle control, growth factor/receptor signal transduction and mobilization of retroelements.

V(D)J recombination is thought to be regulated by changes in the accessibility of target sites, such as modulation of methylation. To test whether demethylation of the kappa locus can activate recombination, we generated two recombinationally active B cell lines in which the gene for maintenance of genomic DNA methylation, Dnmt1 , was flanked with loxP sites. Transduction with a retrovirus expressing both the cre recombinase and green fluorescent protein allowed us to purify recombinationally active cells devoid of methylation. Loss of methylation of the kappa locus was not sufficient to activate recombination, although transcription was activated in one line. It appears that demethylation of the kappa locus is not the rate-limiting step for altering accessibility and thus regulated demethylation does not generate specificity of recombination.

Genome-wide demethylation has been suggested to be a step in carcinogenesis. Evidence for this notion comes from the frequently observed global DNA hypomethylation in tumour cells, and from a recent study suggesting that defects in DNA methylation might contribute to the genomic instability of some colorectal tumour cell lines. DNA hypomethylation has also been associated with abnormal chromosomal structures, as observed in cells from patients with ICF (Immunodeficiency, Centromeric instability and Facial abnormalities) syndrome and in cells treated with the demethylating agent 5-azadeoxycytidine. Here we report that murine embryonic stem cells nullizygous for the major DNA methyltransferase (Dnmt1) gene exhibited significantly elevated mutation rates at both the endogenous hypoxanthine phosphoribosyltransferase (Hprt) gene and an integrated viral thymidine kinase (tk) transgene. Gene deletions were the predominant mutations at both loci. The major cause of the observed tk deletions was either mitotic recombination or chromosomal loss accompanied by duplication of the remaining chromosome. Our results imply an important role for mammalian DNA methylation in maintaining genome stability.

The mouse Xist gene, which is expressed only from the inactive X chromosome, is thought to play a role in the initiation of X inactivation. The 5' end of this gene is fully methylated on the active X chromosome and completely demethylated on the inactive X chromosome, suggesting that DNA methylation may be involved in controlling allele-specific transcription of this gene. To directly investigate the importance of DNA methylation in the control of Xist expression, we have examined its methylation patterns and expression in ES cells and embryos that are deficient in DNA methyltransferase activity. We report here that demethylation of the Xist locus in male mutant embryos induces Xist expression, thus establishing a direct link between demethylation and expression of the Xist gene in the postgastrulation embryo. The transcriptional activity of Xist in undifferentiated ES cells, however, appears to be independent of its methylation status. These results suggest that methylation may only become essential for Xist repression after ES cells have differentiated or after the embryo has undergone gastrulation.

The paternal and maternal genomes are not equivalent and both are required for mammalian development. The difference between the parental genomes is believed to be due to gamete-specific differential modification, a process known as genomic imprinting. The study of transgene methylation has shown that methylation patterns can be inherited in a parent-of-origin-specific manner, suggesting that DNA methylation may play a role in genomic imprinting. The functional significance of DNA methylation in genomic imprinting was strengthened by the recent finding that CpG islands (or sites) in three imprinted genes, H19, insulin-like growth factor 2 (Igf-2), and Igf-2 receptor (Igf-2r), are differentially methylated depending on their parental origin. We have examined the expression of these three imprinted genes in mutant mice that are deficient in DNA methyltransferase activity. We report here that expression of all three genes was affected in mutant embryos: the normally silent paternal allele of the H19 gene was activated, whereas the normally active paternal allele of the Igf-2 gene and the active maternal allele of the Igf-2r gene were repressed. Our results demonstrate that a normal level of DNA methylation is required for controlling differential expression of the paternal and maternal alleles of imprinted genes.

K-ras plays an important role in hematopoiesis. K-ras-deficient mouse embryos die around E12-E13 with severe anemia. In humans, oncogenic mutations in K-ras gene are identified in ~30% of patients with acute myeloid leukemia. We used mouse primary erythroid progenitors as a model system to study the role of K-ras signaling in vivo. Both Epo- and stem cell factor (SCF) - dependent Akt activation are greatly reduced in K-ras-/- fetal liver cells, whereas other cytokine- induced pathways, including Stat5 and p44/p42 MAP kinase, are activated normally. The reduced Akt activation in erythroid progenitors per se leads to delayed erythroid differentiation. Our data identify K-ras as the major regulator for cytokine-dependent Akt activation, which is important for erythroid differentiation in vivo. Overexpression of oncogenic Ras in primary fetal erythroid progenitors led to their continual proliferation and a block in terminal erythroid differentiation. Similarly, we found that primary fetal liver cells expressing oncogenic K-ras from its endogenous locus undergo abnormal proliferation and terminal erythroid differentiation is partially blocked. We are currently investigating the signal transduction pathways activated by this oncogenic K-ras that underlies these cellular phenotypes.

The nucleotide sequence of the B19-Wi isolate of human parvovirus was determined and compared throughout the open reading frames and putative transcription signals with the sequence of the closely related B19-Au isolate. In vitro run off transcription assays, using B19-Wi DNA as the template, indicated that there is a strong promoter between m.u. 5 and 7. Deletion clones show that a region between nt 258 and 321 is necessary for in vitro transcriptional activity. Primer extension studies identified the start site at 31–32 nucleotides downstream of the sequence TATATATA. The strength of this left-hand promoter is unusual among parvovirus promoters characterized to date, and the possibility of an upstream enhancer element is discussed.

Hybrid B19 parvovirus-SV40 origin vectors were transfected into COS-7 cells and replication of these plasmids studied. Plasmids that have a frameshift mutation within the nonstructural gene region replicated to high level (copy number approximately 10,000/transfected cell) although somewhat lower than pSVOd, the SV40 origin vector without B19 sequence (copy number approximately 100,000/transfected cell). However, hybrid B19 parvovirus-SV40 origin vectors that do not contain these frameshift mutations replicated to a much lower level (copy number approximately 1000/transfected cell). Although the hybrid vectors studied replicated at different efficiencies in COS-7 cells, they are transcribed at approximately the same level, resulting in RNA species that are indistinguishable from those seen in B19 virus-infected erythroid bone marrow cells. Western blot analysis demonstrated that the mRNAs are translated into polypeptides of the same size and, in the case of viral structural proteins, in same relative abundance as seen in a B19-infected clinical sample.

The his4-917 mutation of Saccharomyces cerevisiae results from the insertion of the Ty element Ty917 into the regulatory region of the HIS4 gene and renders the cell His-. The hist4-912 delta mutant, which carries a solo delta in the 5'-noncoding region of HIS4, is His+ at 37 degrees C but His- at 23 degrees C. Both these mutations interfere with HIS4 expression at the transcriptional level. The His- phenotype of both insertion mutations is suppressed by mutations at the SPT2 locus. The product of the wild-type SPT2 gene apparently represses HIS4 transcription in these mutant strains; this repression is relieved when the SPT2 gene is destroyed by mutation. The repression of transcription by SPT2 presumably results from an interaction between the SPT2+ gene product and Ty or delta sequences. In this paper, we report the cloning and DNA sequence analysis of the wild-type SPT2 gene and show that the gene is capable of encoding a protein of 333 amino acids in length. In addition, we show that a dominant mutation of the SPT2 gene results from the generation of an ochre codon which is presumed to lead to a shortened SPT2 gene product.

Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency.