Correction: Dissection of NT3 functions in vivo by gene replacement strategy

Development ◽  
2003 ◽  
Vol 130 (1) ◽  
pp. 233-233
Keyword(s):  
Gene Replacement ◽  
Replacement Strategy

scholarly journals Outcomes of Progranulin Gene Therapy in the Retina are Dependent on Time of Delivery

2021 ◽  
Author(s):  
Emilia A. Zin ◽  
Daisy Han ◽  
Jennifer Tran ◽  
Nikolas Morisson-Welch ◽  
Meike Visel ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Quantitative Methods ◽  
Vision Loss ◽  
Neuronal Ceroid Lipofuscinosis ◽  
Cerebellar Atrophy ◽  
Gene Replacement ◽  
Therapeutic Benefit ◽  
Lysosomal Function ◽  
Knockout Mouse Model

AbstractNeuronal ceroid lipofuscinosis (NCL) is a family of neurodegenerative diseases caused by mutations to genes related to lysosomal function. One variant, CNL11, is caused by mutations to the gene encoding the protein progranulin. Primarily secreted by microglia, progranulin regulates neuronal lysosomal function once endocytosed. Absence of progranulin causes cerebellar atrophy, seizures, ataxia, dementia and vision loss. As progranulin gene therapies targeting the brain are developed, it is also advantageous to focus on the retina, as its characteristics are beneficial for gene therapy development: the retina is easily visible through direct imaging, can be assessed through quantitative methods in vivo, requires smaller amounts of AAV and AAV can be administered via a less invasive surgery. In this study we characterize the retinal degeneration in a progranulin knockout mouse model of CLN11 and study the effects of gene replacement at different time points. All mice heterologously expressing progranulin showed reduction in lipofuscin deposits and microglia infiltration. While mice that receive systemic AAV9.2YF-scCAG-PGRN at post-natal day 3 or 4 show a reduction in retina thinning, mice injected intravitreally at months 1 and 6 with 7m8-scCAG-PGRN show no improvement, and mice injected at 12 months of age show increased retinal thinning in comparison to their controls. Thus, delivery of progranulin proves to be time-sensitive, requiring early administration for optimal therapeutic benefit.

An Efficient Site-Directed Mutagenesis Method In Vivo in Escherichia Coli

2012 ◽  
Vol 195-196 ◽  
pp. 407-411
Author(s):  
Mu Qing Qiu
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Recombinant Plasmid ◽  
Genomic Dna ◽  
Gene Replacement ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

In order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knockouted ompR gene was constructed through overlapping PCR, digested by Notand Sal, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR-mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR-mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme

1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687
Author(s):  
C K Barlowe ◽  
D R Appling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Point Mutations ◽  
Gene Replacement ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding

1993 ◽  
Vol 13 (7) ◽  
pp. 4087-4097
Author(s):  
J Wang ◽  
N Suzuki ◽  
Y Nishida ◽  
T Kataoka
Keyword(s):  
Heat Shock ◽  
Adenylyl Cyclase ◽  
Gene Replacement ◽  
Yeast Cells ◽  
Cyclase Activity ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Adenylyl Cyclase Activity

In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

Abstract 603: Effective And Sustained ACE2 Delivery Using Minicircles Technology

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Jan Wysocki ◽  
Philipp K Haber ◽  
Minghao Ye ◽  
Christoph Maier ◽  
Mark J Osborn ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Gene Replacement ◽  
Ang Ii ◽  
Hydrodynamic Approach ◽  
New Gene ◽  
Cloned Cdna ◽  
Dose Dependent ◽  
Delivery Technology

Chronic and sustained amplification of ACE2 activity in vivo has required the development of transgenic mice or the use of viral vectors. Minicircle is a new gene delivery technology which is resistant to gene silencing, and therefore represents an attractive platform for gene replacement strategies in vivo . Here we cloned cDNA of soluble mouse ACE2 into a circular expression cassette and the resulting ACE2 minicircle (MC) was injected to female FVB mice using iv. hydrodynamic approach (10ug or 30ug/mouse). At 3-7d after MC administration, serum ACE2 activity in mice that received 10ug ACE2MC (n=9) was over 100-fold higher than in controls (n=9) (138±48 vs 0.7±0.2 RFU/uL/hr) and in ACE2MC mice (30ug) (n=8) was almost 1000-fold higher than in controls (n=14) (480 ±153 vs 0.5±0.1 RFU/uL/hr, respectively). Mice that received 10 ug ACE2MC were followed for consecutive serum ACE2 activity monitoring, BP measurements and plasma Ang levels. The increase in serum ACE2 activity was sustained until the end of the study (up to 82 days) (Figure). Despite such a marked increase in serum ACE2 activity in ACE2MC mice, conscious SBP was not different from controls (137±8 vs 138±7 mmHg, respectively). At the end of the study, when Ang II was infused acutely (0.2 ug/kg BW i.p.), the increase in plasma Ang II in ACE2MC mice was significantly reduced compared to control mice (915±154 vs 1420±131 fmoL/mL, p<0.05). Mini-circle delivery of ACE2 results in a dose-dependent and sustained long-term increase in serum ACE2 that efficiently degrades plasma Ang II. Extremely high increases in serum ACE2 activity do not reduce BP probably due to activation of non-ACE2 dependent compensatory Ang-hydrolyzing pathways.

scholarly journals Using the Endogenous CRISPR-Cas System of Heliobacterium modesticaldum To Delete the Photochemical Reaction Center Core Subunit Gene

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Patricia L. Baker ◽  
Gregory S. Orf ◽  
Kimberly Kevershan ◽  
Michael E. Pyne ◽  
Taner Bicer ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Reaction Center ◽  
Photochemical Reaction ◽  
Genetic Locus ◽  
Gene Replacement ◽  
Type I ◽  
Content Type ◽  
Heliobacterium Modesticaldum

ABSTRACT In Heliobacterium modesticaldum, as in many Firmicutes, deleting genes by homologous recombination using standard techniques has been extremely difficult. The cells tend to integrate the introduced plasmid into the chromosome by a single recombination event rather than perform the double recombination required to replace the targeted locus. Transformation with a vector containing only a homologous recombination template for replacement of the photochemical reaction center gene pshA produced colonies with multiple genotypes, rather than a clean gene replacement. To address this issue, we required an additional means of selection to force a clean gene replacement. In this study, we report the genetic structure of the type I-A and I-E CRISPR-Cas systems from H. modesticaldum, as well as methods to leverage the type I-A system for genome editing. In silico analysis of the CRISPR spacers revealed a potential consensus protospacer adjacent motif (PAM) required for Cas3 recognition, which was then tested using an in vivo interference assay. Introduction of a homologous recombination plasmid that carried a miniature CRISPR array targeting sequences in pshA (downstream of a naturally occurring PAM sequence) produced nonphototrophic transformants with clean replacements of the pshA gene with ∼80% efficiency. Mutants were confirmed by PCR, sequencing, optical spectroscopy, and growth characteristics. This methodology should be applicable to any genetic locus in the H. modesticaldum genome. IMPORTANCE The heliobacteria are the only phototrophic members of the largely Gram-positive phylum Firmicutes, which contains medically and industrially important members, such as Clostridium difficile and Clostridium acetobutylicum. Heliobacteria are of interest in the study of photosynthesis because their photosynthetic system is unique and the simplest known. Since their discovery in the early 1980s, work on the heliobacteria has been hindered by the lack of a genetic transformation system. The problem of introducing foreign DNA into these bacteria has been recently rectified by our group; however, issues still remained for efficient genome editing. The significance of this work is that we have characterized the endogenous type I CRISPR-Cas system in the heliobacteria and leveraged it to assist in genome editing. Using the CRISPR-Cas system allowed us to isolate transformants with precise replacement of the pshA gene encoding the main subunit of the photochemical reaction center.

scholarly journals TCP1γ Subunit Is Indispensable for Growth and Infectivity of Leishmania donovani

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Shailendra Yadav ◽  
Jitendra Kuldeep ◽  
Mohammad I. Siddiqi ◽  
Neena Goyal
Keyword(s):  
Cell Cycle Progression ◽  
Leishmania Donovani ◽  
Gene Replacement ◽  
Titration Calorimetry ◽  
Content Type ◽  
T Complex ◽  
Complex Protein ◽  
Oligomeric Complex

ABSTRACT T-complex protein-1 (TCP1) is a ubiquitous group II chaperonin and is known to fold various proteins, such as actin and tubulin. In Leishmania donovani, the γ subunit of TCP1 (LdTCP1γ) has been cloned and characterized. It forms a high-molecular-weight homo-oligomeric complex that performs ATP-dependent protein folding. In the present study, we evaluated the essentiality of the LdTCP1γ gene. Gene replacement studies indicated that LdTCP1γ is essential for parasite survival. The LdTCP1γ single-allele-replacement mutants exhibited slowed growth and decreased infectivity in mouse macrophages compared to the growth and infectivity of the wild-type parasites. Modulation of LdTCP1γ expression in promastigotes also modulated cell cycle progression. Suramin, an antitrypanosomal drug, not only inhibited the luciferase refolding activity of the recombinant LdTCP1γ (rLdTCP1γ) homo-oligomeric complex but also exhibited potential antileishmanial efficacy both in vitro and in vivo. The interaction of suramin and LdTCP1γ was further validated by isothermal titration calorimetry. The study suggests LdTCP1γ as a potential drug target and also provides a framework for the development of a new class of drugs.

scholarly journals Induction and role of regulatory CD4+CD25+ T cells in tolerance to the transgene product following hepatic in vivo gene transfer

Blood ◽  
2007 ◽  
Vol 110 (4) ◽  
pp. 1132-1140 ◽  
Author(s):  
Ou Cao ◽  
Eric Dobrzynski ◽  
Lixin Wang ◽  
Sushrusha Nayak ◽  
Bethany Mingle ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Antibody Formation ◽  
Coagulation Factor ◽  
Gene Replacement ◽  
Factor Ix ◽  
In Vivo Gene Transfer

Abstract Gene replacement therapy is complicated by the risk of an immune response against the therapeutic transgene product, which in part is determined by the route of vector administration. Our previous studies demonstrated induction of immune tolerance to coagulation factor IX (FIX) by hepatic adeno-associated viral (AAV) gene transfer. Using a regulatory T-cell (Treg)–deficient model (Rag-2−/− mice transgenic for ovalbumin-specific T-cell receptor DO11.10), we provide first definitive evidence for induction of transgene product-specific CD4+CD25+ Tregs by in vivo gene transfer. Hepatic gene transfer–induced Tregs express FoxP3, GITR, and CTLA4, and suppress CD4+CD25− T cells. Tregs are detected as early as 2 weeks after gene transfer, and increase in frequency in thymus and secondary lymphoid organs during the following 2 months. Similarly, adoptive lymphocyte transfers from mice tolerized to human FIX by hepatic AAV gene transfer indicate induction of CD4+CD25+GITR+ that suppresses antibody formation to FIX. Moreover, in vivo depletion of CD4+CD25+ Tregs leads to antibody formation to the FIX transgene product after hepatic gene transfer, which strongly suggests that these regulatory cells are required for tolerance induction. Our study reveals a crucial role of CD4+CD25+ Tregs in preventing immune responses to the transgene product in gene transfer.

scholarly journals Loss of the F-Actin Binding and Vesicle-Associated Protein Comitin Leads to a Phagocytosis Defect

2002 ◽  
Vol 1 (6) ◽  
pp. 906-914 ◽  
Author(s):  
Thomas Schreiner ◽  
Martina R. Mohrs ◽  
Rosemarie Blau-Wasser ◽  
Alfred von Krempelhuber ◽  
Michael Steinert ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Gene Replacement ◽  
Actin Binding ◽  
Wild Type ◽  
Hyperosmotic Shock ◽  
Latex Beads ◽  
Knockout Mutants ◽  
Mutant Cells

ABSTRACT Comitin is an F-actin binding and membrane-associated protein from Dictyostelium discoideum, which is present on Golgi and vesicle membranes and changes its localization in response to agents affecting the cytoskeleton. To investigate its in vivo functions we have generated knockout mutants by gene replacement. Based on comitin's in vitro functions we examined properties related to vesicular transport and microfilament function. Whereas cell growth, pinocytosis, secretion, chemotaxis, motility, and development were unaltered, comitin-lacking cells were impaired in the early steps of phagocytosis of Saccharomyces cerevisiae particles and of Escherichia coli, whereas uptake of latex beads was unaffected. Furthermore, the lack of comitin positively affected survival of pathogenic bacteria. Mutant cells also showed an altered response to hyperosmotic shock in comparison to the wild type. The redistribution of comitin during hyperosmotic shock in wild-type cells and its presence on early phagosomes suggest a direct involvement of comitin in these processes.

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