Heavy-ion-induced mutations in thegpt delta transgenic mouse: Effect ofp53 gene knockout

2002 ◽  
Vol 40 (3) ◽  
pp. 216-225 ◽  
Author(s):  
Fumio Yatagai ◽  
Toshihiro Kurobe ◽  
Takehiko Nohmi ◽  
Ken-ichi Masumura ◽  
Teruyo Tsukada ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Gene Knockout ◽  
Heavy Ion ◽  
Induced Mutations

Heavy-ion-induced mutations in thegpt delta transgenic mouse: Comparison of mutation spectra induced by heavy-ion, X-ray, and ?-ray radiation

2002 ◽  
Vol 40 (3) ◽  
pp. 207-215 ◽  
Author(s):  
Ken-ichi Masumura ◽  
Kensuke Kuniya ◽  
Toshihiro Kurobe ◽  
Masamichi Fukuoka ◽  
Fumio Yatagai ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Heavy Ion ◽  
Induced Mutations ◽  
X Ray ◽  
Mutation Spectra

scholarly journals Human Desmoglein-2 and Human CD46 Mediate Human Adenovirus Type 55 Infection, but Human Desmoglein-2 Plays the Major Roles

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Ying Feng ◽  
Changhua Yi ◽  
Xinglong Liu ◽  
Linbing Qu ◽  
Wan Su ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Transgenic Mouse ◽  
Gene Knockout ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Host Cells ◽  
Respiratory Pathogen ◽  
Cellular Receptors ◽  
Fiber Protein ◽  
Mouse Lines

ABSTRACT Human adenovirus type 55 (HAdV55) represents an emerging respiratory pathogen and causes severe pneumonia with high fatality in humans. The cellular receptors, which are essential for understanding the infection and pathogenesis of HAdV55, remain unclear. In this study, we found that HAdV55 binding and infection were sharply reduced by disrupting the interaction of viral fiber protein with human desmoglein-2 (hDSG2) but only slightly reduced by disrupting the interaction of viral fiber protein with human CD46 (hCD46). Loss-of-function studies using soluble receptors, blocking antibodies, RNA interference, and gene knockout demonstrated that hDSG2 predominantly mediated HAdV55 infection. Nonpermissive rodent cells became susceptible to HAdV55 infection when hDSG2 or hCD46 was expressed, but hDSG2 mediated more efficient HAd55 infection than hCD46. We generated two transgenic mouse lines that constitutively express either hDSG2 or hCD46. Although nontransgenic mice were resistant to HAdV55 infection, infection with HAdV55 was significantly increased in hDSG2+/+ mice but was much less increased in hCD46+/+ mice. Our findings demonstrate that both hDSG2 and hCD46 are able to mediate HAdV55 infection but hDSG2 plays the major roles. The hDSG2 transgenic mouse can be used as a rodent model for evaluation of HAdV55 vaccine and therapeutics. IMPORTANCE Human adenovirus type 55 (HAdV55) has recently emerged as a highly virulent respiratory pathogen and has been linked to severe and even fatal pneumonia in immunocompetent adults. However, the cellular receptors mediating the entry of HAdV55 into host cells remain unclear, which hinders the establishment of HAdV55-infected animal models and the development of antiviral approaches. In this study, we demonstrated that human desmoglein-2 (hDSG2) plays the major roles during HAdV55 infection. Human CD46 (hCD46) could also mediate the infection of HAdV55, but the efficiency was much lower than for hDSG2. We generated two transgenic mouse lines that express either hDSG2 or hCD46, both of which enabled HAd55 infection in otherwise nontransgenic mice. hDSG2 transgenic mice enabled more efficient HAdV55 infection than hCD46 transgenic mice. Our study adds to our understanding of HAdV55 infection and provides an animal model for evaluating HAdV55 vaccines and therapeutics.

GENE INACTIVATION AND NONMEIOTIC ALLELE INTROGRESSION IN LIVESTOCK SPECIES USING TALENS

2013 ◽  
Vol 25 (1) ◽  
pp. 318 ◽  
Author(s):  
Scott C. Fahrenkrug ◽  
Wenfang Tan ◽  
Simon G. Lillico ◽  
Dana Stverakova ◽  
Chris Proudfoot ◽  
...  
Keyword(s):  
Genetic Improvement ◽  
Gene Editing ◽  
Gene Knockout ◽  
Density Lipoprotein ◽  
Gene Inactivation ◽  
Induced Mutations ◽  
Entire Genome ◽  
Naturally Occurring ◽  
Improvement Programs ◽  
Chromatin Transfer

TALEN-induced double-strand breaks can be used for gene inactivation via repair by non-homologous end joining (NHEJ) or to stimulate homologous recombination (HR). HR can be used to introduce custom genetic modifications or to introgress naturally occurring alleles. We found that over 65% of custom-designed TALENs displayed activity in pig and cattle fibroblasts, with a typical percentage of indel positive chromosomes ranging from 20 to 45%. Isolation of individual clones with mono- and biallelic modifications to targeted loci was extremely efficient (up to 84 and 24% of clones, respectively) and could be accomplished without the aid of selection. Co-transfection of TALENs with a homologous repair template enabled precise insertion of a novel restriction site in nearly 40% of treated cells, with surprising levels of homozygosity. To prove that gene-edited Ossabaw swine cells were suitable for the generation of animals by cloning, we pooled colonies harboring both monoallelic and biallelic TALEN-induced frame-shift mutations in the swine low-density lipoprotein receptor (LDLR) and used them as nuclear donors for chromatin transfer. Pregnancy was established in 7/9 transfers, and 6 pregnancies were carried to term, resulting in the live birth of 18 piglets. Pigs heterozygous and homozygous for TALEN-induced mutations are being investigated as models of familial hypercholesterolemia (FH). We have additionally targeted the same locus for HR using a specified inactivating mutation. Fibroblasts heterozygous and homozygous for a specific 4-bp insertion into LDLR were created by allele introgression and have been cloned by chromatin transfer, demonstrating that gene editing can be used to create precise, swine knock-ins in a single generation. Allele introgression is also critical to livestock genetics, where crossbreeding has been a staple of breeding programs. Although major effect alleles for enhancing productivity and animal welfare have been discovered, the introgression of low-frequency alleles by traditional breeding is slow and inaccurate, involving recombination across the entire genome. The development of gene editing technologies would provide the opportunity to accelerate the genetic improvement in a diversity of livestock breeds. Co-transfection of a TALEN pair with a template containing a specific, naturally occurring allele was effective at the non-meiotic introgression of quantitative traits into the genome of cells from naïve cattle breeds, now being used to create founders by cloning. We will also present progress towards gene conversion by direct injection of livestock embryos. Injection of TALEN mRNA into the cytoplasm of pig and cattle zygotes was capable of inducing gene knockout (KO) in up to 75% of embryos analysed, nearly half of which harbored biallelic modification. We will present alternative strategies for the incorporation of gene editing into livestock genetic improvement programs by either cloning or embryo treatment.

31 PRODUCTION EFFICIENCY OF GENE KNOCKOUT PIGS USING GENOME EDITING AND SOMATIC CELL CLONING

2015 ◽  
Vol 27 (1) ◽  
pp. 108
Author(s):  
H. Matsunari ◽  
M. Watanabe ◽  
K. Nakano ◽  
A. Uchikura ◽  
Y. Asano ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Genome Editing ◽  
Production Efficiency ◽  
Gene Knockout ◽  
Genetically Modified ◽  
Zinc Finger Nucleases ◽  
International Institute ◽  
Induced Mutations ◽  
Transcription Activator

Genome editing technologies have been used as a powerful strategy for the generation of genetically modified pigs. We previously developed genetically modified clone pigs with organogenesis-disabled phenotypes, as well as pigs exhibiting diseases with similar features to those of humans. Here, we report the production efficiency of various gene knockout cloned pigs from somatic cells that were genetically modified using zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN). The ZFN- or TALEN-encoding mRNAs, which targeted 7 autosomal or X-linked genes, were introduced into porcine fetal fibroblast cells using electroporation. Clonal cell populations carrying induced mutations were selected after limiting dilution. The targeted portion of the genes was amplified using PCR, followed by sequencing and mutation analysis. Among the collected knockout cell colonies, cells showing good proliferation and morphology were selected and used for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus-oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. SCNT was performed as reported previously (Matsunari et al. 2008). The cloned embryos were cultured for 7 days in porcine zygote medium (PZM)-5 to assess their developmental ability. Cloned embryos were transplanted into the oviduct or uterus of oestrus-synchronized recipient gilts to evaluate their competence to develop to fetuses or piglets. Cloned embryos reconstructed with 7 types of knockout cells showed equal development to blastocysts compared with those derived from the wild-type cells (54.5–83.3% v. 60.7%). Our data (Table 1) demonstrated that the reconstructed embryos derived from knockout cells could efficiently give rise to cloned offspring regardless of the type of genome editing methodology (i.e. ZFN or TALEN). Table 1.Production efficiency of gene knockout cloned pigs using genome editing This study was supported by JST, ERATO, the Nakauchi Stem Cell and Organ Regeneration Project, JST, CREST, Meiji University International Institute for Bio-Resource Research (MUIIBR), and JSPS KAKENHI Grant Number 26870630.

scholarly journals Transgenic Mouse Technology in Skin Biology: Inducible Gene Knockout in Mice

2014 ◽  
Vol 134 (7) ◽  
pp. 1-4 ◽  
Author(s):  
Christian Günschmann ◽  
Elena Chiticariu ◽  
Bhavuk Garg ◽  
Merve Meliha Hiz ◽  
Yora Mostmans ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Gene Knockout ◽  
Skin Biology ◽  
Inducible Gene

scholarly journals Mutational Consequences of Ciprofloxacin in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 6165-6172 ◽  
Author(s):  
Lisa Yun Song ◽  
Marisa Goff ◽  
Christina Davidian ◽  
Zhiyuan Mao ◽  
Marisa London ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Thymidylate Synthase ◽  
Gene Knockout ◽  
Mutagenic Activity ◽  
Point Mutations ◽  
Base Substitution ◽  
Induced Mutations ◽  
Content Type ◽  
Sublethal Doses ◽  
Substitution Mutations

ABSTRACTWe examined the mutagenic specificity of the widely used antibiotic ciprofloxacin (CPR), which displays weak to moderate mutagenic activity in several bacteria and generates short in-frame deletions inrpoBinStaphylococcus aureus. To determine the spectrum of mutations in a system where any gene knockout would result in a recovered mutant, including frameshifts and both short and long deletions, we examined CPR-induced mutations in the thymidylate synthase-encodingthyAgene. Here, any mutation resulting in loss of thymidylate synthase activity generates trimethoprim (Trm) resistance. We found that deletions and insertions in all three reading frames predominated in the spectrum. They tend to be short deletions and cluster in two regions, one being a GC-rich region with potential extensive secondary structures. We also exploited the well-characterizedrpoB-Rifrsystem inEscherichia colito determine that cells grown in the presence of sublethal doses of CPR not only induced short in-frame deletions inrpoB, but also generated base substitution mutations resulting from induction of the SOS system. Some of the specific point mutations prominent in the spectrum of a strain that overproduces thedinB-encoded Pol IV were also present after growth in CPR. However, these mutations disappeared in CPR-treateddinBmutants, whereas the deletions remained. Moreover, CPR-induced deletions also occurred in a strain lacking all three SOS-induced polymerases. We discuss the implications of these findings for the consequences of overuse of CPR and other antibiotics.

scholarly journals TALEN-Mediated Gene Editing of slc24a5 (Solute Carrier Family 24, Member 5) in Kawakawa, Euthynnus affinis

2021 ◽  
Vol 9 (12) ◽  
pp. 1378
Author(s):  
Dipak Pandey ◽  
Takahiro Matsubara ◽  
Taiju Saito ◽  
Yukinori Kazeto ◽  
Koichiro Gen ◽  
...  
Keyword(s):  
Gene Editing ◽  
Early Stage ◽  
Gene Knockout ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
The Body ◽  
Induced Mutations ◽  
Transcription Activator ◽  
Cell Stage ◽  
Euthynnus Affinis

Transcription activator-like effector (TALE) nucleases (TALENs) mediated gene editing methods are becoming popular and have revealed the staggering complexity of genome control during development. Here, we present a simple and efficient gene knockout using TALENs in kawakawa, Euthynnus affinis, using slc24a5. We examined slc24a5 gene expression and functional differences between two TALENs that hold the TALE scaffolds, +153/+47 and +136/+63 and target slc24a5. Developmental changes in slc24a5 transcripts were seen in early-stage embryos by real-time PCR; slc24a5 expression was first detected 48 h post fertilization (hpf), which increased dramatically at 72 hpf. Four TALENs, 47- and 63-type of two different target loci (A and B), respectively, were constructed using Platinum TALEN and evaluated in vitro by a single-strand annealing (SSA) assay. TALEN activities were further evaluated in vivo by injecting TALEN mRNAs in the two-cell stage of the zygote. Most of the TALEN-induced mutants showed mosaic patterns in the retinal pigment epithelium (RPE) and fewer melanin pigments on the body at 72 hpf and later when compared to the control, implying the gene’s association with melanin pigment formation. A heteroduplex mobility assay (HMA) and the genome sequence further confirmed the TALEN-induced mutations of substitution, insertion, and deletion at an endogenous locus.

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