Rapid and precise genotyping of transgene zygosity in mice using an allele-specific method.

Precise determination of transgene zygosity is essential for use of transgenic mice in research. Because integration loci of transgenes are usually unknown due to their random insertion, assessment of transgene zygosity remains a challenge. Current zygosity genotyping methods (progeny testing, qPCR, and NGS-computational biology analysis) are time consuming, prone to error or technically challenging. Here, we developed a novel method to determine transgene zygosity requiring no knowledge of transgene insertion loci. This method applies allele-specific restriction enzyme digestion of PCR products (RE/PCR) to rapidly and reliably quantify transgene zygosity. We demonstrate the applicability of this method to three transgenic strains of mice (Atm TgC3001L, Nes-Cre, and Syn1-Cre) harboring a unique restriction enzyme site on either the transgene or its homologous sequence in the mouse genome. This method is as accurate as the gold standard of progeny testing but requires 2 d instead of a month or more. It is also exceedingly more accurate than the most commonly used approach of qPCR quantification. Our novel method represents a significant technical advance in determining transgene zygosities in mice.

Expression and production of pigment epithelium-derived factor (PEDF) and PEDF receptor variants from mammalian and bacterial cells.

Human SERPINF1 gene codes for pigment epithelium-derived factor (PEDF), a secreted glycoprotein and member of the SERPIN superfamily. To obtain large amounts of recombinant PEDF proteins, we subcloned the coding sequence of human SERPINF1 mutated versions into the pCEP4 vector and generated stably transfected HEK.Ebna cells. The cells produced and secreted recombinant PEDF proteins into the culturing media. The recombinant PEDF proteins were purified by ion-exchange column chromatography and milligram amounts of highly purified protein were recovered. PEDF has affinity for PEDF-receptor (PEDF-R), a membrane-linked lipase encoded by the PNPLA2 gene. Recombinant PEDF-R truncated versions were obtained from Escherichia coli containing expression vectors with human PNPLA2 cDNAs with 3'end deletions and by induction with isopropyl ß-d-1-thiogalactopyranoside. The bacterially derived PEDF-R proteins in insoluble inclusion bodies were solubilized with urea and purified by cation-exchange column chromatography. C-terminally truncated PEDF-R versions containing the ligand binding region retained the ability to bind PEDF. The data demonstrate that mammalian-derived recombinant PEDF and bacterially derived recombinant PEDF-R can be produced and purified in large amounts for further use in structural and biological studies.

Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R).

Purpose: To examine the contribution of pigment epithelium-derived factor receptor (PEDF-R) to the phagocytosis process. Previously, we identified PEDF-R, the protein encoded by the PNPLA2 gene, as a phospholipase A2 in the retinal pigment epithelium (RPE). During phagocytosis, RPE cells ingest abundant phospholipids and protein in the form of photoreceptor outer segment (POS) tips, which are then hydrolyzed. The role of PEDF-R in RPE phagocytosis is not known. Methods: Mice in which PNPLA2 was conditionally knocked out (cKO) in the RPE were generated. Mouse RPE/choroid explants were cultured. Human ARPE-19 cells were transfected with siPNPLA2 silencing duplexes. POSs were isolated from bovine retinas. The phospholipase A2 inhibitor bromoenol lactone was used. Transmission electron microscopy, immunofluorescence, lipid labeling, pulse-chase experiments, western blots, and free fatty acid and ß-hydroxybutyrate assays were performed. Results: The RPE of the cKO mice accumulated lipids, as well as more abundant and larger rhodopsin particles, compared to littermate controls. Upon POS exposure, RPE explants from cKO mice released less ß-hydroxybutyrate compared to controls. After POS ingestion during phagocytosis, rhodopsin degradation was stalled both in cells treated with bromoenol lactone and in PNPLA2-knocked-down cells relative to their corresponding controls. Phospholipase A2 inhibition lowered ß-hydroxybutyrate release from phagocytic RPE cells. PNPLA2 knockdown also resulted in a decline in fatty acids and ß-hydroxybutyrate release from phagocytic RPE cells. Conclusions: PEDF-R downregulation delayed POS digestion during phagocytosis. The findings imply that the efficiency of RPE phagocytosis depends on PEDF-R, thus identifying a novel contribution of this protein to POS degradation in the RPE.

PEDF deficiency increases the susceptibility of rd10 mice to retinal degeneration.

The SERPINF1 gene encodes pigment epithelium-derived factor (PEDF), a member of the serpin superfamily with neurotrophic and antiangiogenic properties in the retina. We hypothesized that absence of PEDF would lead to increased stress-associated retinal degeneration in Serpinf1 null mice. Accordingly, using a Serpinf1 null mouse model, we investigated the impact of PEDF absence on retinal morphology, and susceptibility to induced and inherited retinal degeneration. We studied the pattern of Serpinf1 expression in the mouse retina layers. PEDF protein was detected by western blotting. Transmission electron microscopy was performed on mouse retina. Serpinf1 null mice and wild type littermates were injected with NaIO3 (30 mg/kg body weight) intraperitonially. At post-injection day 1, 3, 4, 6 and 8 mice were euthanized, and eyes were enucleated. Serpinf1 null and rd10 double mutant mice were generated and their eyes enucleated at different time points from post-natal day 15 to post-natal day 28. Enucleated eyes were processed for hematoxylin and eosin staining and histopathological evaluations. We found that Serpinf1 was expressed in the retinal pigment epithelium, in the inner nuclear layer and in the ganglion cell layer, but undetectable in the outer nuclear layer of wild type mice. Plasma PEDF protein levels were undetectable in Serpinf1 null animals. RPE atrophy and retinal thinning were observed in NaIO3-treated wild type mice that progressed with time post-injection. NaIO3-treated Serpinf1 null mice showed comparatively better retinal morphology than wild type mice at day 4 post-injection. However, the absence of PEDF in Serpinf1 null x rd10 mice increased the susceptibility to retinal degeneration relative to that of rd10 mice. We concluded that histopathological evaluation of retinas lacking PEDF showed that removal of the Serpinf1 gene may activate PEDF-independent compensatory mechanisms to protect the retina against oxidative stress, while it increases the susceptibility to degenerate the retina in inherited retinal degeneration models.

PEDF peptides promote photoreceptor survival in rd10 retina models.

The purpose of the study is to evaluate the protective properties of PEDF peptide fragments on rd10 mouse models of retinal degeneration ex vivo. Human recombinant PEDF and synthetic peptides were used. Rd10 retinal explants as well as wild-type retinal explants treated with zaprinast to mimic the rd10 photoreceptor cell death were employed. PEDF protein was intravitreally administered into rd10 mice. Outer nuclear layer thickness measurements in retinal sections, TUNEL labeling in retinal explants, western blots and immunofluorescence with retinal samples were performed. PEDF protein levels in the RPE of rd10 mice decreased with age (P15 - P25). Levels of PEDF receptor PEDF-R declined in the photoreceptor inner segments from rd10 relative to wild-type mice at P25. PEDF administration increased the outer nuclear layer thickness of rd10 retinas in vivo and decreased the number of TUNEL+ nuclei of photoreceptors in rd10 retinal explant cultures, both relative to untreated controls. Peptides containing the PEDF neurotrophic region decreased the number of TUNEL+ photoreceptors in both rd10 and zaprinast-induced cell death ex vivo models, while peptides without the neurotrophic region and/or lacking affinity for PEDF-R were ineffective in protecting photoreceptors. Thus, retinal explants are a valuable system to evaluate PEDF activity. Short peptides with the photoreceptor-protective property of PEDF may prove useful for the development of therapeutic agents for photoreceptor protection in retinal degenerations.

Pigment Epithelium-Derived Factor, a Protective Factor for Photoreceptors in Vivo.

Pigment epithelium-derived factor (PEDF) is a natural protein of the retina with demonstrable neurotrophic properties, found in the interphotoreceptor matrix in intimate contact with photoreceptors. This review summarizes the effects of PEDF on photoreceptors in several animal models of retinal degeneration.

Small Retinoprotective Peptides Reveal a Receptor-binding Region on Pigment Epithelium-derived Factor.

The cytoprotective effects of pigment epithelium-derived factor (PEDF) require interactions between an as of a yet undefined region with a distinct ectodomain on the PEDF receptor (PEDF-R). Here we characterized the area in PEDF that interacts with PEDF-R to promote photoreceptor survival. Molecular docking studies suggested that the ligand binding site of PEDF-R interacts with the neurotrophic region of PEDF (44-mer, positions 78-121). Binding assays demonstrated that PEDF-R bound the 44-mer peptide. Moreover, peptide P1 from the PEDF-R ectodomain had affinity for the 44-mer and a shorter fragment within it, 17-mer (positions 98-114). Single residue substitutions to alanine along the 17-mer sequence were designed and tested for binding and biological activity. Altered 17-mer[R99A] did not bind to the P1 peptide, whereas 17-mer[H105A] had higher affinity than the unmodified 17-mer. Peptides 17-mer, 17-mer[H105A], and 44-mer exhibited cytoprotective effects in cultured retina R28 cells. Intravitreal injections of these peptides and PEDF in the rd1 mouse model of retinal degeneration decreased the numbers of dying photoreceptors, 17-mer[H105A] being most effective. The blocking peptide P1 hindered their protective effects both in retina cells and in vivo. Thus, in addition to demonstrating that the region composed of positions 98-114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects, the findings reveal distinct small PEDF fragments with neurotrophic effects on photoreceptors.

CtIP-mediated resection is essential for viability and can operate independently of BRCA1.

Homologous recombination (HR) is initiated by DNA end resection, a process in which stretches of single-strand DNA (ssDNA) are generated and used for homology search. Factors implicated in resection include nucleases MRE11, EXO1, and DNA2, which process DNA ends into 3' ssDNA overhangs; helicases such as BLM, which unwind DNA; and other proteins such as BRCA1 and CtIP whose functions remain unclear. CDK-mediated phosphorylation of CtIP on T847 is required to promote resection, whereas CDK-dependent phosphorylation of CtIP-S327 is required for interaction with BRCA1. Here, we provide evidence that CtIP functions independently of BRCA1 in promoting DSB end resection. First, using mouse models expressing S327A or T847A mutant CtIP as a sole species, and B cells deficient in CtIP, we show that loss of the CtIP-BRCA1 interaction does not detectably affect resection, maintenance of genomic stability or viability, whereas T847 is essential for these functions. Second, although loss of 53BP1 rescues the embryonic lethality and HR defects in BRCA1-deficient mice, it does not restore viability or genome integrity in CtIP(-/-) mice. Third, the increased resection afforded by loss of 53BP1 and the rescue of BRCA1-deficiency depend on CtIP but not EXO1. Finally, the sensitivity of BRCA1-deficient cells to poly ADP ribose polymerase (PARP) inhibition is partially rescued by the phospho-mimicking mutant CtIP (CtIP-T847E). Thus, in contrast to BRCA1, CtIP has indispensable roles in promoting resection and embryonic development.

DRAGO (KIAA0247), a new DNA damage-responsive, p53-inducible gene that cooperates with p53 as oncosuppressor. [Corrected].

BACKGROUND: p53 influences genomic stability, apoptosis, autophagy, response to stress, and DNA damage. New p53-target genes could elucidate mechanisms through which p53 controls cell integrity and response to damage. METHODS: DRAGO (drug-activated gene overexpressed, KIAA0247) was characterized by bioinformatics methods as well as by real-time polymerase chain reaction, chromatin immunoprecipitation and luciferase assays, time-lapse microscopy, and cell viability assays. Transgenic mice (94 p53(-/-) and 107 p53(+/-) mice on a C57BL/6J background) were used to assess DRAGO activity in vivo. Survival analyses were performed using Kaplan-Meier curves and the Mantel-Haenszel test. All statistical tests were two-sided. RESULTS: We identified DRAGO as a new p53-responsive gene induced upon treatment with DNA-damaging agents. DRAGO is highly conserved, and its ectopic overexpression resulted in growth suppression and cell death. DRAGO(-/-) mice are viable without macroscopic alterations. However, in p53(-/-) or p53(+/-) mice, the deletion of both DRAGO alleles statistically significantly accelerated tumor development and shortened lifespan compared with p53(-/-) or p53(+/-) mice bearing wild-type DRAGO alleles (p53(-/-), DRAGO(-/-) mice: hazard ratio [HR] = 3.25, 95% confidence interval [CI] = 1.7 to 6.1, P < .001; p53(+/-), DRAGO(-/-) mice: HR = 2.35, 95% CI = 1.3 to 4.0, P < .001; both groups compared with DRAGO(+/+) counterparts). DRAGO mRNA levels were statistically significantly reduced in advanced-stage, compared with early-stage, ovarian tumors, but no mutations were found in several human tumors. We show that DRAGO expression is regulated both at transcriptional-through p53 (and p73) and methylation-dependent control-and post-transcriptional levels by miRNAs. CONCLUSIONS: DRAGO represents a new p53-dependent gene highly regulated in human cells and whose expression cooperates with p53 in tumor suppressor functions.

Role of environment and sex differences in the development of autoimmune diseases: a roundtable meeting report.

Autoimmune diseases (ADs) impose substantial health and financial burdens in the United States and in many parts of the world. Women are disproportionately affected by many of these disorders, which often contribute to lifelong disabilities. While the number of patients with some ADs appears to be rising, the complexities of conducting epidemiological studies prevent a thorough understanding of the prevalence and incidence of these various conditions. Research on environmental influences of these illnesses is limited, although they are generally hypothesized to result from the interaction of environmental agents in genetically susceptible individuals. Further, there is little known regarding the role of sex and gender in the environmentally influenced mechanisms leading to the development of AD. To address these issues, particularly the roles of environment and sex and gender in ADs and the factors that contribute to the rise in ADs, the Society for Women's Health Research convened an interdisciplinary roundtable of experts from academia, medicine, and government agencies to share their expertise, address knowledge gaps in research, and propose future research recommendations.

53BP1 mediates productive and mutagenic DNA repair through distinct phosphoprotein interactions.

The DNA damage response (DDR) protein 53BP1 protects DNA ends from excessive resection in G1, and thereby favors repair by nonhomologous end-joining (NHEJ) as opposed to homologous recombination (HR). During S phase, BRCA1 antagonizes 53BP1 to promote HR. The pro-NHEJ and antirecombinase functions of 53BP1 are mediated in part by RIF1, the only known factor that requires 53BP1 phosphorylation for its recruitment to double-strand breaks (DSBs). Here, we show that a 53BP1 phosphomutant, 53BP18A, comprising alanine substitutions of the eight most N-terminal S/TQ phosphorylation sites, mimics 53BP1 deficiency by restoring genome stability in BRCA1-deficient cells yet behaves like wild-type 53BP1 with respect to immunoglobulin class switch recombination (CSR). 53BP18A recruits RIF1 but fails to recruit the DDR protein PTIP to DSBs, and disruption of PTIP phenocopies 53BP18A. We conclude that 53BP1 promotes productive CSR and suppresses mutagenic DNA repair through distinct phosphodependent interactions with RIF1 and PTIP.

Mechanism of DNA resection during intrachromosomal recombination and immunoglobulin class switching.

DNA double-strand breaks (DSBs) are byproducts of normal cellular metabolism and obligate intermediates in antigen receptor diversification reactions. These lesions are potentially dangerous because they can lead to deletion of genetic material or chromosome translocation. The chromatin-binding protein 53BP1 and the histone variant H2AX are required for efficient class switch (CSR) and V(D)J recombination in part because they protect DNA ends from resection and thereby favor nonhomologous end joining (NHEJ). Here, we examine the mechanism of DNA end resection in primary B cells. We find that resection depends on both CtBP-interacting protein (CtIP, Rbbp8) and exonuclease 1 (Exo1). Inhibition of CtIP partially rescues the CSR defect in 53BP1- and H2AX-deficient lymphocytes, as does interference with the RecQ helicases Bloom (Blm) and Werner (Wrn). We conclude that CtIP, Exo1, and RecQ helicases contribute to the metabolism of DNA ends during DSB repair in B lymphocytes and that minimizing resection favors efficient CSR.

53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks.

Defective DNA repair by homologous recombination (HR) is thought to be a major contributor to tumorigenesis in individuals carrying Brca1 mutations. Here, we show that DNA breaks in Brca1-deficient cells are aberrantly joined into complex chromosome rearrangements by a process dependent on the nonhomologous end-joining (NHEJ) factors 53BP1 and DNA ligase 4. Loss of 53BP1 alleviates hypersensitivity of Brca1 mutant cells to PARP inhibition and restores error-free repair by HR. Mechanistically, 53BP1 deletion promotes ATM-dependent processing of broken DNA ends to produce recombinogenic single-stranded DNA competent for HR. In contrast, Lig4 deficiency does not rescue the HR defect in Brca1 mutant cells but prevents the joining of chromatid breaks into chromosome rearrangements. Our results illustrate that HR and NHEJ compete to process DNA breaks that arise during DNA replication and that shifting the balance between these pathways can be exploited to selectively protect or kill cells harboring Brca1 mutations.

PRL-3 phosphatase is implicated in ovarian cancer growth.

PURPOSE: The PRL-3 phosphatase has been found expressed at higher levels in metastasis than in primary tumors of patients with colorectal cancer. In the present study, we evaluated the expression of PRL-3 in ovarian cancer tissue and its role in ovarian cancer cell growth. EXPERIMENTAL DESIGN: PRL-3 phosphatase expression was evaluated in 84 ovarian tumor samples. PRL-3 expression has been knocked down using specific small interfering RNAs to determine its role in ovarian cancer cell growth in vitro. RESULTS: In ovarian cancers, PRL-3 expression correlates with disease progression, being higher in advanced (stage III) than in early (stage I) tumors. In situ measurements of PRL-3 expression showed that it was confined to the epithelial neoplastic cells. The molecular mechanism underlying PRL-3 overexpression in ovarian cancers is independent from amplification of the corresponding genomic locus. Ovarian cancer cells growing in culture have high levels of expression of this phosphatase. PRL-3-specific knockdown using small interfering RNA severely impaired the growth of cells without affecting the expression of the closely related homologue PRL-1. Intriguingly, the growth of human colon carcinoma cells expressing lower levels of the PRL-3 was not affected by the PRL-3 knockdown. CONCLUSIONS: Altogether, these results show that PRL-3 expression is associated with ovarian cancer progression and point to a key role for this phosphatase in the control of ovarian cancer cells growth. This strongly suggests that PRL-3 should be considered as a target for the discovery of new anticancer agents to be tested against this malignancy.