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1.  Simple Monitoring of Gene Targeting Efficiency in Human Somatic Cell Lines Using the PIGA Gene 
PLoS ONE  2012;7(10):e47389.
Gene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvement of this technology and its application to a broad range of cell lines. In this study, we utilized phosphatidylinositol glycan anchor biosynthesis class A (PIGA), a gene essential for the synthesis of glycosylphosphatidyl inositol (GPI) anchors, as a reporter of gene targeting events in human somatic cell lines. Targeted disruption of PIGA was quantitatively detected with FLAER, a reagent that specifically binds to GPI anchors. Using this PIGA-based reporter system, we successfully detected adeno-associated virus (AAV)-mediated gene targeting events both with and without promoter-trap enrichment of gene-targeted cell population. The PIGA-based reporter system was also capable of reproducing previous findings that an AAV-mediated gene targeting achieves a remarkably higher ratio of homologous versus random integration (H/R ratio) of targeting vectors than a plasmid-mediated gene targeting. The PIGA-based system also detected an approximately 2-fold increase in the H/R ratio achieved by a small negative selection cassette introduced at the end of the AAV-based targeting vector with a promoter-trap system. Thus, our PIGA-based system is useful for monitoring AAV-mediated gene targeting and will assist in improving gene targeting technology in human somatic cell lines.
PMCID: PMC3466256  PMID: 23056640
2.  Impaired growth and elevated Fas receptor expression in PIGA+ stem cells in primary paroxysmal nocturnal hemoglobinuria 
Journal of Clinical Investigation  2000;106(5):689-696.
The genetic defect underlying paroxysmal nocturnal hemoglobinuria (PNH) has been shown to reside in PIGA, a gene that encodes an element required for the first step in glycophosphatidylinositol anchor assembly. Why PIGA-mutated cells are able to expand in PNH marrow, however, is as yet unclear. To address this question, we compared the growth of affected CD59–CD34+ and unaffected CD59+CD34+ cells from patients with that of normal CD59+CD34+ cells in liquid culture. One hundred FACS-sorted cells were added per well into microtiter plates, and after 11 days at 37°C the progeny were counted and were analyzed for their differentiation pattern. We found that CD59–CD34+ cells from PNH patients proliferated to levels approaching those of normal cells, but that CD59+CD34+ cells from the patients gave rise to 20- to 140-fold fewer cells. Prior to sorting, the patients’ CD59– and CD59+CD34+ cells were equivalent with respect to early differentiation markers, and following culture, the CD45 differentiation patterns were identical to those of control CD34+ cells. Further analyses of the unsorted CD59+CD34+ population, however, showed elevated levels of Fas receptor. Addition of agonist anti-Fas mAb to cultures reduced the CD59+CD34+ cell yield by up to 78% but had a minimal effect on the CD59–CD34+ cells, whereas antagonist anti-Fas mAb enhanced the yield by up to 250%. These results suggest that expansion of PIGA-mutated cells in PNH marrow is due to a growth defect in nonmutated cells, and that greater susceptibility to apoptosis is one factor involved in the growth impairment.
PMCID: PMC381282  PMID: 10974022
3.  A mutation in a functional Sp1 binding site of the telomerase RNA gene (hTERC) promoter in a patient with Paroxysmal Nocturnal Haemoglobinuria 
Mutations in the gene coding for the RNA component of telomerase, hTERC, have been found in autosomal dominant dyskeratosis congenita (DC) and aplastic anemia. Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal blood disorder associated with aplastic anemia and characterized by the presence of one or more clones of blood cells lacking glycosylphosphatidylinositol (GPI) anchored proteins due to a somatic mutation in the PIGA gene.
We searched for mutations in DNA extracted from PNH patients by amplification of the hTERC gene and denaturing high performance liquid chromatography (dHPLC). After a mutation was found in a potential transcription factor binding site in one patient electrophoretic mobility shift assays were used to detect binding of transcription factors to that site. The effect of the mutation on the function of the promoter was tested by transient transfection constructs in which the promoter is used to drive a reporter gene.
Here we report the finding of a novel promoter mutation (-99C->G) in the hTERC gene in a patient with PNH. The mutation disrupts an Sp1 binding site and destroys its ability to bind Sp1. Transient transfection assays show that mutations in this hTERC site including C-99G cause either up- or down-regulation of promoter activity and suggest that the site regulates core promoter activity in a context dependent manner in cancer cells.
These data are the first report of an hTERC promoter mutation from a patient sample which can modulate core promoter activity in vitro, raising the possibility that the mutation may affect the transcription of the gene in hematopoietic stem cells in vivo, and that dysregulation of telomerase may play a role in the development of bone marrow failure and the evolution of PNH clones.
PMCID: PMC442127  PMID: 15212690
4.  Murine embryonic stem cells without pig-a gene activity are competent for hematopoiesis with the PNH phenotype but not for clonal expansion. 
Journal of Clinical Investigation  1997;100(5):1028-1036.
Paroxysmal nocturnal hemoglobinuria (PNH) develops in patients who have had a somatic mutation in the X-linked PIG-A gene in a hematopoietic stem cell; as a result, a proportion of blood cells are deficient in all glycosyl phosphatidylinositol (GPI)-anchored proteins. Although the PIG-A mutation explains the phenotype of PNH cells, the mechanism enabling the PNH stem cell to expand is not clear. To examine this growth behavior, and to investigate the role of GPI-linked proteins in hematopoietic differentiation, we have inactivated the pig-a gene by homologous recombination in mouse embryonic stem (ES) cells. In mouse chimeras, pig-a- ES cells were able to contribute to hematopoiesis and to differentiate into mature red cells, granulocytes, and lymphocytes with the PNH phenotype. The proportion of PNH red cells was substantial in the fetus, but decreased rapidly after birth. Likewise, PNH granulocytes could only be demonstrated in the young mouse. In contrast, the percentage of lymphocytes deficient in GPI-linked proteins was more stable. In vitro, pig-a- ES cells were able to form pig-a- embryoid bodies and to undergo hematopoietic (erythroid and myeloid) differentiation. The number and the percentage of pig-a- embryoid bodies with hematopoietic differentiation, however, were significantly lower when compared with wild-type embryoid bodies. Our findings demonstrate that murine ES cells with a nonfunctional pig-a gene are competent for hematopoiesis, and give rise to blood cells with the PNH phenotype. pig-a inactivation on its own, however, does not confer a proliferative advantage to the hematopoietic stem cell. This provides direct evidence for the notion that some additional factor(s) are needed for the expansion of the mutant clone in patients with PNH.
PMCID: PMC508277  PMID: 9276719
5.  GPI-anchor synthesis is indispensable for the germline development of the nematode Caenorhabditis elegans 
Molecular Biology of the Cell  2012;23(6):982-995.
Twenty-four Caenorhabditis elegans genes are involved in GPI-anchor synthesis. Based on the isolation of a deletion allele of the PIGA gene mediating the first step of GPI-anchor synthesis, GPI-anchor synthesis in somatic gonads and/or in germline is shown to be indispensable for the normal development of oocytes and eggs.
Glycosylphosphatidylinositol (GPI)-anchor attachment is one of the most common posttranslational protein modifications. Using the nematode Caenorhabditis elegans, we determined that GPI-anchored proteins are present in germline cells and distal tip cells, which are essential for the maintenance of the germline stem cell niche. We identified 24 C. elegans genes involved in GPI-anchor synthesis. Inhibition of various steps of GPI-anchor synthesis by RNA interference or gene knockout resulted in abnormal development of oocytes and early embryos, and both lethal and sterile phenotypes were observed. The piga-1 gene (orthologue of human PIGA) codes for the catalytic subunit of the phosphatidylinositol N-acetylglucosaminyltransferase complex, which catalyzes the first step of GPI-anchor synthesis. We isolated piga-1–knockout worms and found that GPI-anchor synthesis is indispensable for the maintenance of mitotic germline cell number. The knockout worms displayed 100% lethality, with decreased mitotic germline cells and abnormal eggshell formation. Using cell-specific rescue of the null allele, we showed that expression of piga-1 in somatic gonads and/or in germline is sufficient for normal embryonic development and the maintenance of the germline mitotic cells. These results clearly demonstrate that GPI-anchor synthesis is indispensable for germline formation and for normal development of oocytes and eggs.
PMCID: PMC3302757  PMID: 22298425
6.  Paroxysmal Nocturnal Hemoglobinuria from Bench to Bedside 
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disease that presents with protean manifestations. Clinical and laboratory investigation over the past 25 years have uncovered most of the basic science underpinnings of PNH and have led to the development of a highly effective targeted therapy. PNH originates from a multipotent hematopoietic stem cell (HSC) that acquires a somatic mutation in a gene called phosphatidylinositol glycan anchor biosynthesis, class A (PIG-A). The PIG-A gene is required for the first step in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Failure to synthesize GPI anchors leads to an absence of all proteins that utilize GPI to attach to the plasma membrane. Two GPI-anchor proteins, CD55 and CD59, are complement regulatory proteins; their absence on the surface of PNH cells leads to complement-mediated hemolysis. The release of free hemoglobin leads to scavenging of nitric oxide and contributes to many clinical manifestations, including esophageal spasm, fatigue, and possibly thrombosis. Aerolysin, a pore-forming toxin, binds GPI anchored proteins and kills normal cells, but not PNH cells. A fluorescinated aerolysin variant (FLAER) binds GPI-anchor and serves as a novel reagent diagnosing PNH. Eculizumab, a humanized monoclonal antibody against C5, is the first effective drug therapy for PNH.
PMCID: PMC3128433  PMID: 21707954
7.  Paroxysmal nocturnal hemoglobinuria: pathophysiology, natural history and treatment options in the era of biological agents 
Biologics : Targets & Therapy  2008;2(2):205-222.
Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal non-malignant hematological disease characterized by the expansion of hematopoietic stem cells (HSCs) and progeny mature cells, whose surfaces lack all the proteins linked through the glycosyl-phosphatidyl inositol anchor. This defect arises from an acquired somatic mutation in the X-linked phosphatidylinositol glycan class A gene, with subsequent clonal expansion of the mutated HSCs as a result of a concomitant, likely immune-mediated, selective pressure. The disease is characterized by complement-mediated chronic intravascular hemolysis, resulting in hemolytic anemia and hemosiderinuria; capricious exacerbations lead to recurrent gross hemoglobinuria. Additional cardinal manifestations of PNH are a variable degree of bone marrow failure and an intrinsic propensity to thromboembolic events. The disease is markedly invalidating, with chronic symptoms requiring supportive therapy – usually including periodical transfusions; possible life-threatening complications may also ensue. The biology of PNH has been progressively elucidated in the past few years, but therapeutic strategies remained unsatisfactory for decades, the only exception being stem cell transplantation, which is restricted to selected patients and retains significant morbidity and mortality. Recently, a biological agent to treat PNH has been developed – the terminal complement inhibitor eculizumab – which has been tested in a number of clinical trials, with exciting results. All the data from worldwide clinical trials confirm that eculizumab radically modifies the symptoms, the biology, and the natural history of PNH, strongly improving the quality of life of PNH patients.
PMCID: PMC2721357  PMID: 19707355
paroxysmal nocturnal hemoglobinuria; GPI-AP; PIG-A; complement; eculizumab
8.  Generation of Glycosylphosphatidylinositol Anchor Protein-Deficient Blood Cells From Human Induced Pluripotent Stem Cells 
Stem Cells Translational Medicine  2013;2(11):819-829.
Using PIG-A gene targeting and an inducible PIG-A expression system, this study established a conditional PIG-A knockout model in human induced pluripotent stem cells that allowed for the production of glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient blood cells. This conditional PIG-A knockout model should be a valuable tool for studying the importance of GPI-APs in hematopoiesis and human development.
PIG-A is an X-linked gene required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; thus, PIG-A mutant cells have a deficiency or absence of all GPI-anchored proteins (GPI-APs). Acquired mutations in hematopoietic stem cells result in the disease paroxysmal nocturnal hemoglobinuria, and hypomorphic germline PIG-A mutations lead to severe developmental abnormalities, seizures, and early death. Human induced pluripotent stem cells (iPSCs) can differentiate into cell types derived from all three germ layers, providing a novel developmental system for modeling human diseases. Using PIG-A gene targeting and an inducible PIG-A expression system, we have established, for the first time, a conditional PIG-A knockout model in human iPSCs that allows for the production of GPI-AP-deficient blood cells. PIG-A-null iPSCs were unable to generate hematopoietic cells or any cells expressing the CD34 marker and were defective in generating mesodermal cells expressing KDR/VEGFR2 (kinase insert domain receptor) and CD56 markers. In addition, PIG-A-null iPSCs had a block in embryonic development prior to mesoderm differentiation that appears to be due to defective signaling through bone morphogenetic protein 4. However, early inducible PIG-A transgene expression allowed for the generation of GPI-AP-deficient blood cells. This conditional PIG-A knockout model should be a valuable tool for studying the importance of GPI-APs in hematopoiesis and human development.
PMCID: PMC3808197  PMID: 24113066
Human iPSCs; GPI-anchor protein; Hematopoiesis; PIG-A gene knockout
9.  A method for the generation of conditional gene repair mutations in mice 
Nucleic Acids Research  2001;29(3):e10.
Conditional gene repair mutations in the mouse can assist in cell lineage analyses and provide a valuable complement to conditional gene inactivation strategies. We present a method for the generation of conditional gene repair mutations that employs a loxP-flanked (floxed) selectable marker and transcriptional/translational stop cassette (neostop) located within the first intron of a target gene. In the absence of Cre recombinase, expression of the targeted allele is suppressed generating a null allele, while in the presence of Cre, excision of neostop restores expression to wild-type levels. To test this strategy, we have generated a conditional gene repair allele of the mouse Huntington’s disease gene homolog (Hdh). Insertion of neostop within the Hdh intron 1 generated a null allele and mice homozygous for this allele resembled nullizygous Hdh mutants and died after embryonic day 8.5. In the presence of a cre transgene expressed ubiquitously early in development, excision of neostop restored Hdh expression and rescued the early embryonic lethality. A simple modification of this strategy that permits the generation of conventional gene knockout, conditional gene knockout and conditional gene repair alleles using one targeting construct is discussed.
PMCID: PMC30411  PMID: 11160912
10.  Silencing Inhibits Cre-Mediated Recombination of the Z/AP and Z/EG Reporters in Adult Cells 
PLoS ONE  2009;4(5):e5435.
The Cre-loxP system has been used to enable tissue specific activation, inactivation and mutation of many genes in vivo and has thereby greatly facilitated the genetic dissection of several cellular and developmental processes. In such studies, Cre-reporter strains, which carry a Cre-activated marker gene, are frequently utilized to validate the expression profile of Cre transgenes, to act as a surrogate marker for excision of a second allele, and to irreversibly label cells for lineage tracing experiments.
Principal Findings
We have studied three commonly used Cre-reporter strains, Z/AP, Z/EG and R26R-EYFP and have demonstrated that although each reporter can be reliably activated by Cre during early development, exposure to Cre in adult hematopoietic cells results in a much lower frequency of marker-positive cells in the Z/AP or Z/EG strains than in the R26R-EYFP strain. In marker negative cells derived from the Z/AP and Z/EG strains, the transgenic promoter is methylated and Cre-mediated recombination of the locus is inhibited.
These results show that the efficiency of Cre-mediated recombination is not only dependent on the genomic context of a given loxP-flanked sequence, but also on stochastic epigenetic mechanisms underlying transgene variegation. Furthermore, our data highlights the potential shortcomings of utilizing the Z/AP and Z/EG reporters as surrogate markers of excision or in lineage tracing experiments.
PMCID: PMC2672169  PMID: 19415111
11.  β-Catenin Is Dispensable for Hematopoiesis and Lymphopoiesis 
β-catenin–mediated Wnt signaling has been suggested to be critically involved in hematopoietic stem cell maintenance and development of T and B cells in the immune system. Unexpectedly, here we report that inducible Cre-loxP–mediated inactivation of the β-catenin gene in bone marrow progenitors does not impair their ability to self-renew and reconstitute all hematopoietic lineages (myeloid, erythroid, and lymphoid), even in competitive mixed chimeras. In addition, both thymocyte survival and antigen-induced proliferation of peripheral T cells is β-catenin independent. In contrast to earlier reports, these data exclude an essential role for β-catenin during hematopoiesis and lymphopoiesis.
PMCID: PMC2211763  PMID: 14718516
Wnt signaling; T cells; B cells; development; gene targeting
12.  Non-parallel recombination limits Cre-LoxP-based reporters as precise indicators of conditional genetic manipulation 
Genesis (New York, N.Y. : 2000)  2013;51(6):436-442.
Cre/LoxP-mediated recombination allows for conditional gene activation or inactivation. When combined with an independent lineage-tracing reporter allele, this technique traces the lineage of presumptive genetically modified Cre-expressing cells. Several studies have suggested that floxed alleles have differential sensitivities to Cre-mediated recombination, which raises concerns regarding utilization of common Cre-reporters to monitor recombination of other floxed loci of interest. Here, we directly investigate the recombination correlation, at cellular resolution, between several floxed alleles induced by Cre-expressing mouse lines. The recombination correlation between different reporter alleles varied greatly in otherwise genetically identical cell types. The chromosomal location of floxed alleles, distance between LoxP sites, sequences flanking the LoxP sites, and the level of Cre activity per cell all likely contribute to observed variations in recombination correlation. These findings directly demonstrate that, due to non-parallel recombination events, commonly available Cre reporter mice cannot be reliably utilized, in all cases, to trace cells that have DNA recombination in independent-target floxed alleles, and that careful validation of recombination correlations are required for proper interpretation of studies designed to trace the lineage of genetically modified populations, especially in mosaic situations.
PMCID: PMC3696028  PMID: 23441020
Mosaic analysis; lineage tracing; cell autonomous; Cre detection; non-parallel recombination
13.  The transcription factors Stat5a/b are not required for islet development but modulate pancreatic β-cell physiology upon aging 
Biochimica et biophysica acta  2007;1773(9):1455-1461.
In insulinoma cell lines proliferation and insulin gene transcription are stimulated by growth hormone and prolactin, which convey their signals through the transcription factors Stat5a and 5b (referred to as Stat5). However, the contribution of Stat5 to the physiology of β-cells in vivo could not be assessed directly since Stat5-null mice die perinataly. To explore the physiological role of Stat5 in the mouse, the corresponding gene locus targeted with loxP sites was inactivated in β-cells using two lines of Cre recombinase expressing transgenic mice. While the RIP-Cre transgene is active in pancreatic β-cells and the hypothalamus, the Pdx1-Cre transgene is active in precursor cells of the endocrine and exocrine pancreas. Mice carrying two floxed Stat5 alleles and a RIP-Cre transgene developed mild obesity, were hyperglycemic and exhibited impaired glucose tolerance. Since RIP-Cre transgenic mice by themselves display some glucose intolerance, the significance of these data is unclear. In contrast, mice, in which the Stat5 locus had been deleted with the Pdx1-Cre transgene, developed functional islets and were glucose tolerant. Mild glucose intolerance occurred with age. We conclude that Stat5 is not essential for islet development but may modulate β-cell function.
PMCID: PMC2695665  PMID: 17599554
Stat5; Rip-Cre; Pdx1-Cre; Islets of Langerhans; β-cells; glucose homeostasis
Genesis (New York, N.Y. : 2000)  2009;47(12):789-792.
The albCre transgene, having Cre recombinase driven by the serum albumin (alb) gene promoter, is commonly used to generate adult mice having reliable hepatocyte-specific recombination of loxP-flanked (“floxed”) alleles. Based on previous studies, it has been unclear whether albCre transgenes are also reliable in fetal and juvenile mice. Perinatal liver undergoes a dynamic transition from being predominantly hematopoietic to predominantly hepatic. We evaluated Cre activity during this transition in albCre mice using a sensitive two-color fluorescent reporter system. From fetal through adult stages, in situ patterns of Cre-dependent recombination of the reporter closely matched expression of endogenous Alb mRNA or protein, indicating most or all hepatocytes, including those in fetal and juvenile livers, had expressed Cre and recombined the reporter. Our results indicate the albCre transgene is effective at converting simple floxed alleles in fetal and neonatal mice and is an appropriate tool for studies on hepatocyte development.
PMCID: PMC2828742  PMID: 19830819
Albumin-Cre; conditional allele; liver-specific knockout; liver development; hepatocyte; fetal albumin expression
15.  Generation of a Conditional Null Allele of Jumonji 
Genesis (New York, N.Y. : 2000)  2006;44(9):407-411.
The jumonji (jmj) gene plays important roles in multiple organ development in mouse, including cardiovascular development. Since JMJ is expressed widely during mouse development, it is essential that conditional knockout approaches be employed to ablate JMJ in a tissue-specific manner to identify the cell lineage specific roles of JMJ. In this report, we describe the establishment of a jmj conditional null allele in mice by generating a loxP-flanked (floxed) jmj allele, which allows the in vivo ablation of jmj via Cre recombinase-mediated deletion. Gene targeting was used to introduce loxP sites flanking exon 3 of the jmj allele to mouse embryonic stem cells. Our results indicate that the jmj floxed allele converts to a null allele in a heart-specific manner when embryos homozygous for the floxed jmj allele and carrying the α-myosin heavy chain promoter-Cre transgene were analyzed by Southern and Northern blot analyses. Therefore, this mouse line harboring the conditional jmj null allele will provide a valuable tool for deciphering the tissue and cell lineage specific roles of JMJ.
PMCID: PMC2002517  PMID: 16900512
Jumonji (Jarid 2); Cre-loxP technology; conditional knockout; mouse development; heart
16.  Targeted expression of Cre recombinase to adipose tissue of transgenic mice directs adipose-specific excision of loxP-flanked gene segments. 
Nucleic Acids Research  1997;25(12):2543-2545.
Functional analysis of mammalian genes relies, in part, on targeted mutations generated by homologous recombination in mice. We have developed a strategy for adipose-specific inactivation of loxP-floxed gene segments. Transgenic mice have been established that express Cre recombinase under the control of the adipose-specific aP2 enhancer/promoter. Crossing of the aP2/ Cre mice with any loxP-floxed gene will facilitate its functional analysis in adipose tissue.
PMCID: PMC146759  PMID: 9171115
17.  Simultaneous deletion of floxed genes mediated by CaMKIIα-Cre in the brain and in male germ cells: application to conditional and conventional disruption of Goα 
The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) promoter limits expression to specific regions of the forebrain and thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKIIα-Cre can be utilized for simultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26+/stop-lacZ::CaMKIIα-Cre+/Cre mice generated by crossing CaMKIIα-Cre+/Cre mice with floxed ROSA26 lacZ reporter (Rosa26+/stop-lacZ) mice exhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% of the offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate that recombination can occur in the germ cells of male Rosa26+/stop-lacZ::CaMKIIα-Cre+/Cre mice. Similarly, when double transgenic Gnao+/f::CaMKIIα-Cre+/Cre mice carrying a floxed Go-alpha gene (Gnaof/f) were backcrossed to wild-type females, approximately 22% of the offspring carried the disrupted allele (GnaoΔ) without inheriting the Cre transgene. The GnaoΔ/Δ mice closely resembled conventional Go-alpha knockout mice (Gnao−/−) with respect to impairment of their behavior. Thus, we conclude that CaMKIIα-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brain and for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKIIα-Cre mice as breeding pairs.
PMCID: PMC3972788  PMID: 24787734
brain; Cre; CaMKII alpha; Gnao; testis
18.  Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. 
Nucleic Acids Research  1999;27(22):4324-4327.
Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ER(T), a fusion protein between a mutated ligand binding domain of the human estrogen receptor (ER) and the Cre recombinase, the activity of which can be induced by 4-hydroxy-tamoxifen (OHT), but not natural ER ligands. We have recently characterized a new ligand-dependent recombinase, Cre-ER(T2), which was approximately 4-fold more efficiently induced by OHT than Cre-ER(T) in cultured cells. In order to compare the in vivo efficiency of these two ligand-inducible recombinases to generate temporally-controlled somatic mutations, we have engineered transgenic mice expressing a LoxP-flanked (floxed) transgene reporter and either Cre-ER(T) or Cre-ER(T2) under the control of the bovine keratin 5 promoter that is specifically active in the epidermis basal cell layer. No background recombinase activity could be detected, while recombination was induced in basal keratinocytes upon OHT administration. Interestingly, a dose-response study showed that Cre-ER(T2) was approximately 10-fold more sensitive to OHT induction than Cre-ER(T).
PMCID: PMC148712  PMID: 10536138
19.  Cre reconstitution allows for DNA recombination selectively in dual-marker-expressing cells in transgenic mice 
Nucleic Acids Research  2007;35(19):e126.
Cre/LoxP-based DNA recombination has been used to introduce desired DNA rearrangements in various organisms, having for example, greatly assisted genetic analyses in mice. For most applications, single gene promoters are used to drive Cre production for conditional gene activation/inactivation or lineage-tracing experiments. Such a manipulation introduces Cre in all cells in which the utilized promoter is active. To overcome the limited selectivity of single promoters for cell-type-specific recombination, we have explored the ‘dual promoter combinatorial control’ of Cre activity, so that Cre activity could be restricted to cells that express dual protein markers. We efficiently reconstituted Cre activity from two modified, inactive Cre fragments. Cre re-association was greatly enhanced by fusing the Cre fragments separately to peptides that can form a tight antiparallel leucine zipper. The co-expressed Cre fusion fragments showed substantial activity in cultured cells. As proof of principle of the utility of this technique in vivo for manipulating genes specifically in dual-marker-positive cells, we expressed each inactive Cre fragments in transgenic mice via individual promoters. Result showed the effective reconstitution of Cre activates LoxP recombination in the co-expressing cells.
PMCID: PMC2095822  PMID: 17893102
20.  Mutation analysis of the PIG-A gene in Korean patients with paroxysmal nocturnal haemoglobinuria 
Journal of Clinical Pathology  2002;55(6):410-413.
Aim: Paroxysmal nocturnal haemoglobinuria (PNH) is caused by deficient biosynthesis of the glycosylphosphatidylinositol (GPI) anchor in haemopoietic stem cells. Mutation of the phosphatidylinositol glycan class A (PIG-A) gene, an X linked gene that participates in the first step of GPI anchor biosynthesis, is responsible for PNH. The characteristics of somatic mutation of the PIG-A gene in Korean patients with PNH were studied.
Methods: Twenty four patients with PNH were selected. Ham tests and sucrose haemolysis tests were carried out on all patients. The expression of CD59 in erythrocytes and granulocytes was investigated in 14 and five patients, respectively, to confirm the diagnosis. Dideoxy fingerprinting (ddF) was used to screen mutations, and direct sequencing of DNA was performed to characterise the mutations.
Results: Gene mutation was detected in 12 of the 24 patients. The other 12 patients were negative in ddF screening. Ten new mutations and two known mutations were detected. The mutations consisted of five deletions, six substitutions, and one insertion. These mutations resulted in six premature terminations, three abnormal splicings, one missense mutation in exon 2, and two nonsense mutations. Two patients with venous thrombosis showed mutations in exon 3 only. Substitution mutations were seen in six patients and frameshift mutations in the other six.
Conclusions: There were 10 new mutations among the 12 mutations in the Korean patients with PNH and the characteristics of the mutations varied, with no significant hot spots in sites or types.
PMCID: PMC1769670  PMID: 12037021
paroxysmal nocturnal haemoglobinuria; mutation; phosphatidylinositol glycan class A (PIG-A) gene; glycosylphosphatidylinositol anchor; dideoxy fingerprinting
21.  Increased soluble urokinase plasminogen activator receptor (suPAR) is associated with thrombosis and inhibition of plasmin generation in paroxysmal nocturnal hemoglobinuria (PNH) patients 
Experimental hematology  2008;36(12):1616-1624.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired genetic disorder of the bone marrow that produces intravascular hemolysis, proclivity to venous thrombosis, and hematopoietic failure. Mutation in the PIG-A gene of a hematopoietic stem cell abrogates synthesis of glycosylphosphoinositol (GPI) anchors and expression of all GPI-anchored proteins on the surface of progeny erythrocytes, leukocytes, and platelets. Urokinase plasminogen activator receptor (uPAR), a GPI-linked protein expressed on neutrophils, mediates endogenous thrombolysis through a urokinase-dependent mechanism. Here we show that membrane GPI-anchored uPAR is decreased or absent on granulocytes and platelets of patients with PNH, while soluble uPAR (suPAR) levels are increased in patients’ plasma. Serum suPAR concentrations correlated with the number of GPI-negative neutrophils and were highest in patients who later develop thrombosis. In vitro, suPAR is released from PNH hematopoietic cells and from platelets upon activation, suggesting that these cells are the probable source of plasma suPAR in the absence of GPI anchor synthesis and trafficking of uPAR to the cell membrane. In vitro, the addition of recombinant suPAR results in a dose-dependent decrease in the activity of single-chain urokinase. We hypothesized that suPAR, prevents the interaction of urokinase with membrane-anchored uPAR on residual normal cells.
PMCID: PMC3417356  PMID: 18954937
22.  Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors 
BMC Neuroscience  2004;5:4.
Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV) and lentiviral (LV) vectors into discrete regions of the forebrain.
Recombinant AAV-Cre, AAV-GFP (green fluorescent protein) and LV-Cre-EGFP (enhanced GFP) were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection.
AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.
PMCID: PMC343275  PMID: 15005815
23.  Acquired Deficiency of A20 Results in Rapid Apoptosis, Systemic Inflammation, and Abnormal Hematopoietic Stem Cell Function 
PLoS ONE  2014;9(1):e87425.
A20 is a negative regulator of NF-κB, and mutational loss of A20 expression is involved in the pathogenesis of autoimmune diseases and B-cell lymphomas. To clarify the role of A20 in adult hematopoiesis, we generated conditional A20 knockout mice (A20flox/flox) and crossed them with Mx–1Cre (MxCre+) and ERT2Cre (ERT2Cre+) transgenic mice in which Cre is inducibly activated by endogenous interferon and exogenous tamoxifen, respectively. A20flox/flox MxCre+ (A20Mx) mice spontaneously exhibited myeloid proliferation, B cell apoptosis, and anemia with overproduction of pro-inflammatory cytokines. Bone marrow transplantation demonstrated that these changes were caused by hematopoietic cells. NF-κB was constitutively activated in A20Mx hematopoietic stem cells (HSCs), which caused enhanced cell cycle entry and impaired repopulating ability. Tamoxifen stimulation of A20flox/flox ERT2Cre+ (A20ERT2) mice induced fulminant apoptosis and subsequent myeloproliferation, lymphocytopenia, and progressive anemia with excessive production of pro-inflammatory cytokines, as observed in A20Mx mice. These results demonstrate that A20 plays essential roles in the homeostasis of adult hematopoiesis by preventing apoptosis and inflammation. Our findings provide insights into the mechanism underlying A20 dysfunction and human diseases in which A20 expression is impaired.
PMCID: PMC3909109  PMID: 24498102
24.  Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts 
The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.
PMCID: PMC3157869  PMID: 20864926
25.  Frequency of paroxysmal nocturnal hemoglobinuria in patients attended in Belém, Pará, Brazil 
Paroxysmal nocturnal hemoglobinuria is a hematological disease with complex physiopathology. It is genetically characterized by a somatic mutation in the PIG-A gene (phosphatidylinositol glycan anchor biosynthesis, class A), in which the best known antigens are DAF (decay accelerating factor or CD55) and MIRL (membrane inhibitor of reactive lysis or CD59).
To determine the frequency of paroxysmal nocturnal hemoglobinuria in patients attended at the HEMOPA foundation from November 2008 to July 2009.
Thirty patients, with ages ranging from two to 79 years old and suspected of having paroxysmal nocturnal hemoglobinuria were examined. All patients were immunophenotyped by flow cytometry for the CD5, CD59, CD16 and CD45 antigens.
Paroxysmal nocturnal hemoglobinuria was identified in nine of the thirty patients investigated. Another 3 cases had inconclusive results with CD59-negative labeling only for neutrophils. The highest frequency of paroxysmal nocturnal hemoglobinuria patients (7/9) and inconclusive cases (2/3) were between 19 years old and 48 years old, with a median of 28 years.
These results show the importance of flow cytometry to identify cases in which patients are deficient in only one antigen (CD59).
PMCID: PMC3521433  PMID: 23284241
Immunophenotyping; Flow cytometry; Paroxysmal nocturnal hemoglobinuria; Diagnosis; Antigen, CD55; Antigen, CD59

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