Publications by authors named "Leslie O Goodwin"

10 Publications

  • Page 1 of 1

Sequentially inducible mouse models reveal that Npm1 mutation causes malignant transformation of Dnmt3a-mutant clonal hematopoiesis.

Leukemia 2019 07 28;33(7):1635-1649. Epub 2019 Jan 28.

The Jackson Laboratory, Bar Harbor, ME, USA.

Clonal hematopoiesis (CH) is a common aging-associated condition with increased risk of hematologic malignancy. Knowledge of the mechanisms driving evolution from CH to overt malignancy has been hampered by a lack of in vivo models that orthogonally activate mutant alleles. Here, we develop independently regulatable mutations in DNA methyltransferase 3A (Dnmt3a) and nucleophosmin 1 (Npm1), observed in human CH and AML, respectively. We find Dnmt3a mutation expands hematopoietic stem and multipotent progenitor cells (HSC/MPPs), modeling CH. Induction of mutant Npm1 after development of Dnmt3a-mutant CH causes progression to myeloproliferative disorder (MPD), and more aggressive MPD is observed with longer latency between mutations. MPDs uniformly progress to acute myeloid leukemia (AML) following transplant, accompanied by a decrease in HSC/MPPs and an increase in myeloid-restricted progenitors, the latter of which propagate AML in tertiary recipient mice. At a molecular level, progression of CH to MPD is accompanied by selection for mutations activating Ras/Raf/MAPK signaling. Progression to AML is characterized by additional oncogenic signaling mutations (Ptpn11, Pik3r1, Flt3) and/or mutations in epigenetic regulators (Hdac1, Idh1, Arid1a). Together, our study demonstrates that Npm1 mutation drives evolution of Dnmt3a-mutant CH to AML and rate of disease progression is accelerated with longer latency of CH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41375-018-0368-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609470PMC
July 2019

Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis.

Genome Res 2019 03 18;29(3):494-505. Epub 2019 Jan 18.

The Jackson Laboratory, Bar Harbor, Maine 04609, USA.

Transgenesis has been a mainstay of mouse genetics for over 30 yr, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or an essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported, due in part to limitations in the discovery tools. Targeted locus amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.233866.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396414PMC
March 2019

Viable Mice with Extensive Gene Humanization (25-kbp) Created Using Embryonic Stem Cell/Blastocyst and CRISPR/Zygote Injection Approaches.

Sci Rep 2018 10 9;8(1):15028. Epub 2018 Oct 9.

Genetic Resource Science, The Jackson Laboratory, Bar Harbor, ME, USA.

Here, we describe an expansion of the typical DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus by zygotic injection. Driving our efforts was the desire to create a whole animal model that would replace 17 kilobase pairs (kbp) of the mouse Bcl2l11 gene with the corresponding 25-kbp segment of human BCL2L11, including a conditionally removable segment (2.9-kbp) of intron 2, a cryptic human exon immediately 3' of this, and a native human exon some 20 kbp downstream. Using two methods, we first carried out the replacement by employing a combination of bacterial artificial chromosome recombineering, classic embryonic stem cell (ESC) targeting, dual selection, and recombinase-driven cassette removal (ESC/Blastocyst Approach). Using a unique second method, we employed the same vector (devoid of its selectable marker cassettes), microinjecting it along with redundant single guide RNAs (sgRNAs) and Cas9 mRNA into mouse zygotes (CRISPR/Zygote Approach). In both instances, we were able to achieve humanization of Bcl2l11 to the extent designed, remove all selection cassettes, and demonstrate the functionality of the conditionally removable, loxP-flanked, 2.9-kbp intronic segment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-33408-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177426PMC
October 2018

CRISPRtools: a flexible computational platform for performing CRISPR/Cas9 experiments in the mouse.

Mamm Genome 2017 Aug 9;28(7-8):283-290. Epub 2017 Mar 9.

The Jackson Laboratory, 600 Main St., Bar Harbor, ME, 04609, USA.

Genome editing using the CRISPR/Cas9 RNA-guided endonuclease system has rapidly become a driving force for discovery in modern biomedical research. This simple yet elegant system has been widely used to generate both loss-of-function alleles and precision knock-in mutations using single-stranded donor oligonucleotides. Our CRISPRtools platform supports both of these applications in order to facilitate the use of CRISPR/Cas9. While there are several tools that facilitate CRISPR/Cas9 design and screen for potential off-target sites, the process is typically performed sequentially on single genes, limiting scalability for large-scale programs. Here, the design principle underlying gene ablation is based upon using paired guides flanking a critical region/exon of interest to create deletions. Guide pairs are rank ordered based upon published efficiency scores and off-target analyses, and reported in a concise format for downstream implementation. The exon deletion strategy simplifies characterization of founder animals and is the strategy employed for the majority of knockouts in the mouse. In proof-of-principle experiments, the effectiveness of this approach is demonstrated using microinjection and electroporation to introduce CRISPR/Cas9 components into mouse zygotes to delete critical exons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00335-017-9681-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591755PMC
August 2017

Modulation of rat intestinal nuclear factor NF-kappaB by gum arabic.

Dig Dis Sci 2008 Jan 8;53(1):80-7. Epub 2007 May 8.

The Feinstein Institute for Medical Research, North Shore-Long Island Jewish Health System, Manhasset, NY 11030, USA.

The objective of this study was to test the hypothesis that in an animal model of cathartic-induce intestinal dysfunction the proabsorptive effects of gum arabic (GA) could be associated with modulation of nuclear factor-kappaB (NF-kappaB) and with reduction of the inflammatory response caused by cathartics, as evidenced by intestinal mucosa cytokine production and gene expression. Juvenile male rats were given a phenolphthalein-magnesium citrate solution for 6 days, by itself or supplemented with either 10 or 20 g L(-1) GA, as a sole source of fluid. The controls given were tap water alone or with added 20 g L(-1) GA. The animals were euthanized and small-intestinal mucosa nuclear fractions and RNA were isolated. NF-kappaB p65 activity was highest after administration of cathartics, lowest in controls, and intermediate in GA-treated rats. Mucosal IL-1beta was overexpressed in tissues from cathartic-treated rats and from rats given high-GA solutions. Gene-array analysis revealed a complex pattern of gene regulation by cathartics which selectively upregulated several subfamilies of cytochrome P-450 family 2 genes. Co-administration of GA did not block this effect. These findings suggest that local anti-inflammatory effects on the small intestine could be obtained by administration of a nonabsorbable proteoglycan such as GA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10620-007-9826-0DOI Listing
January 2008

Voltage-dependent calcium channels in mammalian spermatozoa revisited.

Front Biosci 2007 Jan 1;12:1420-49. Epub 2007 Jan 1.

Center for Oncology and Cell Biology, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, New York 11030, USA.

The last few years have seen an explosion in the number of voltage-dependent ion channel sequences detected in sperm and testes. The complex structural paradigm of these channels is now known to include a pore-forming alpha1 subunit(s) whose electrophysiological properties are modulated by an intracellular beta subunit, a disulfide-linked complex of a membrane-spanning delta subunit with an extracellular alpha2 subunit, and a transmembrane gamma subunit. Many of these are alternatively spliced. Furthermore, the known number of genes coding each subtype has expanded significantly (10 alpha1, 4 beta, 4 alpha2delta, 8 gamma). Recently, the CatSper gene family has been characterized based on similarity to the voltage-dependent calcium channel alpha1 subunit. From among this multiplicity, a wide cross-section is active in sperm, including many splice variants. For example, expression of the various alpha1 subunits appears strictly localized in discrete domains of mature sperm, and seems to control distinct physiological roles such as cellular signaling pathways. These include alpha1 alternative splicing variants that are regulated by ions passed by channels in developing sperm. Various combinations of ion channel sequence variants have been studies in research models and in a variety of human diseases, including male infertility. For example, rats that are genetically resistant to testes damage by lead seem to respond to lead ions by increasing alpha1 alternative splicing. In contrast, in varicocele-associated male infertility, the outcome from surgical correction correlates with suppression of alpha1 alternative splicing, Ion channel blockers remain attractive model contraceptive drugs because of their ability to modulate cholesterol levels. However, the large number of sperm ion channel variants shared with other cell types make ion channels less attractive targets for male contraceptive development than a few years ago. In this review, the genetics, structure and function of voltage-dependent calcium channels and related CatSper molecules will be discussed, and several practical clinical applications associated with these channels will be reported.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2741/2158DOI Listing
January 2007

Role of glutamine depletion in directing tissue-specific nutrient stress responses to L-asparaginase.

J Biol Chem 2006 Oct 24;281(42):31222-33. Epub 2006 Aug 24.

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Evansville, Indiana 47712, USA.

L-asparaginase is important in the induction regimen for treating acute lymphoblastic leukemia. Cytotoxic complications are clinically significant problems lacking mechanistic insight. To reveal tissue-specific molecular responses to this drug, mice were administered asparaginase from either Escherichia coli (clinically used) or Wolinella succinogenes (novel, glutaminase-free form). Both enzymes abolished serum asparagine, but only the E. coli form reduced circulating glutamine. E. coli asparaginase reduced protein synthesis in liver and spleen but not pancreas via increased phosphorylation of the translation factor eIF2. In contrast, treatment with Wolinella caused no untoward changes in protein synthesis in any tissue examined. Treating mice deleted for the eIF2 kinase, GCN2, with the E. coli enzyme showed eIF2 phosphorylation to be GCN2-dependent, but only initially. Furthermore, although eIF2 phosphorylation was not increased in the pancreas or by Wolinella asparaginase, expression of the amino acid stress response genes, asparagine synthetase and CHOP/GADD153, increased as a result of both enzymes, even in tissues demonstrating no change in eIF2 phosphorylation. Finally, signaling downstream of the mammalian target of rapamycin kinase was repressed in liver and pancreas by E. coli but not Wolinella asparaginase. These data demonstrate that the nutrient stress response to asparaginase is tissue-specific and exacerbated by glutamine depletion. Importantly, increased expression of asparagine synthetase and CHOP does not require eIF2 phosphorylation, signifying alternate or auxiliary means of inducing gene expression under conditions of amino acid depletion in the whole animal.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M604511200DOI Listing
October 2006

Gene expression profiling reveals a signaling role of glutathione in redox regulation.

Proc Natl Acad Sci U S A 2005 Sep 19;102(39):13998-4003. Epub 2005 Sep 19.

Laboratory of Neuroimmunology "Mario Negri," Institute for Pharmacological Research, 20157 Milan, Italy.

Proteins can form reversible mixed disulfides with glutathione (GSH). It has been hypothesized that protein glutathionylation may represent a mechanism of redox regulation, in a fashion similar to that mediated by protein phosphorylation. We investigated whether GSH has a signaling role in the response of HL60 cells to hydrogen peroxide (H2O2), in addition to its obvious antioxidant role. We identified early changes in gene expression induced at different times by H2O2 treatment, under conditions that increase protein glutathionylation and minimal toxicity. We then investigated the effect of prior GSH depletion by buthionine sulfoximine and diethylmaleate on this response. The analysis revealed 2,016 genes regulated by H2O2. Of these, 215 genes showed GSH-dependent expression changes, classifiable into four clusters displaying down- or up-regulation by H2O2, either potentiated or inhibited by GSH depletion. The modulation of 20 selected genes was validated by real-time RT-PCR. The biological process categories overrepresented in the largest cluster (genes whose up-regulation was inhibited by GSH depletion) were NF-kappaB activation, transcription, and DNA methylation. This cluster also included several cytokine and chemokine ligands and receptors, the redox regulator thioredoxin interacting protein, and the histone deacetylase sirtuin. The cluster of genes whose up-regulation was potentiated by GSH depletion included two HSPs (HSP40 and HSP70) and the AP-1 transcription factor components Fos and FosB. This work demonstrates that GSH, in addition to its antioxidant and protective function against oxidative stress, has a specific signaling role in redox regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0504398102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1236550PMC
September 2005

Deletions in L-type calcium channel alpha1 subunit testicular transcripts correlate with testicular cadmium and apoptosis in infertile men with varicoceles.

Fertil Steril 2005 Mar;83(3):622-34

Fertility Research Laboratories, North Shore-Long Island Jewish Research Institute, North Shore University Hospital, Manhasset, New York, USA.

Objective: To identify and understand predictors of successful varicocelectomy.

Design: Examination of testicular L-type voltage-dependent calcium channel (L-VDCC) mRNAs and proteins in testis biopsies and comparison of presence and absence of various mRNAs with testicular cadmium levels, with apoptosis, and with sperm count change after varicocelectomy.

Setting: University clinical urology practice and research laboratories.

Patient(s): Infertile men with varicocele (left varicocele only, n = 18; bilateral varicoceles, n = 26) and controls (men with obstructive azoospermia undergoing testicular sperm extraction before intracytoplasmic sperm injection; n = 7).

Intervention(s): Left testis biopsies by percutaneous needle aspiration biopsy. Varicocele repair by subinguinal approach.

Main Outcome Measure(s): Calcium channel mRNA sequence by reverse transcription-polymerase chain reaction and amplicon analysis; calcium channel protein distribution by immunocytochemistry; cadmium levels by atomic absorption and apoptosis by deoxynucleotidyl transferase labeling; and sperm counts in the ejaculate before and after varicocelectomy.

Result(s): Calcium channel mRNAs are polymorphic in human testis biopsies from different men. Proteins from sequence-deleted exons 7 and/or 8 localize to germ cell membranes. Expression of undeleted L-type calcium channel mRNAs correlates with normal testes cadmium and increased sperm count after varicocelectomy. Apoptosis is lower in such cases.

Conclusion(s): Expression of normal testicular L-VDCC sequence in exons 7-8 predicts postvaricocelectomy sperm count increase. Deletions may alter calcium channel function and affect testicular cadmium and apoptosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fertnstert.2004.07.976DOI Listing
March 2005