Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)

Mortensen, Richard M.

doi:10.1002/0471142301.ns0430s21

Cited by 7 publications

(6 citation statements)

References 21 publications

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“…We generated mice that lacked the MGST1 gene by conventional methodology [21] , [22] , [23] . Upon breeding heterozygous mice, the offspring did not contain any homozygous null mice.…”

Section: Resultsmentioning

confidence: 99%

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MGST1, a GSH transferase/peroxidase essential for development and hematopoietic stem cell differentiation

et al. 2018

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We show for the first time that, in contrast to other glutathione transferases and peroxidases, deletion of microsomal glutathione transferase 1 (MGST1) in mice is embryonic lethal. To elucidate why, we used zebrafish development as a model system and found that knockdown of MGST1 produced impaired hematopoiesis. We show that MGST1 is expressed early during zebrafish development and plays an important role in hematopoiesis. High expression of MGST1 was detected in regions of active hematopoiesis and co-expressed with markers for hematopoietic stem cells. Further, morpholino-mediated knock-down of MGST1 led to a significant reduction of differentiated hematopoietic cells both from the myeloid and the lymphoid lineages. In fact, hemoglobin was virtually absent in the knock-down fish as revealed by diaminofluorene staining. The impact of MGST1 on hematopoiesis was also shown in hematopoietic stem/progenitor cells (HSPC) isolated from mice, where it was expressed at high levels. Upon promoting HSPC differentiation, lentiviral shRNA MGST1 knockdown significantly reduced differentiated, dedicated cells of the hematopoietic system. Further, MGST1 knockdown resulted in a significant lowering of mitochondrial metabolism and an induction of glycolytic enzymes, energetic states closely coupled to HSPC dynamics. Thus, the non-selenium, glutathione dependent redox regulatory enzyme MGST1 is crucial for embryonic development and for hematopoiesis in vertebrates.

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“…We generated mice that lacked the MGST1 gene by conventional methodology [21] , [22] , [23] . Upon breeding heterozygous mice, the offspring did not contain any homozygous null mice.…”

Section: Resultsmentioning

confidence: 99%

“…Production and breeding of mice missing the MGST1 gene was by conventional methods [21] , [22] , [23] by the UCSD Transgenic Animal Core Services. Mice were maintained under standard 12-h light/12-h dark cycle with access to standard rodent food and given water ad libitum.…”

Section: Methodsmentioning

confidence: 99%

MGST1, a GSH transferase/peroxidase essential for development and hematopoietic stem cell differentiation

et al. 2018

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“…The targeting vector that was used for the creation of the mutant mouse line is shown in Figure 1, and was produced by modifying pNTK [Mortensen, 2008] as previously described [Zubenko and Hughes, 2011]. The genomes of 78 mice from the mutant mouse colony established at the Jackson Laboratory were evaluated for the presence of three bacterial regions of the targeting vector by PCR amplification using the primer pairs listed in Table I.…”

Section: Resultsmentioning

confidence: 99%

Purification, surface modification of coal ash silica and its potential application in rubber composites

Mahmood

Khan

et al. 2010

J of Applied Polymer Sci

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This article reports the purification and surface modification of coal ash silica and afterwards its utilization as reinforcing filler in solution-styrene-butadiene rubber/butadiene rubber (S-SBR/BR). The coal ash silica free of unwanted metal ions/mineral oxides was obtained using phosphoric acid. The chemical composition of the purified coal ash silica in comparison to impure coal ash indicates the presence of characteristic hydroxyl functional group at 3440 cm À1 and an increase in the oxygen content as determined with the help of Fourier Transform Infrared and x-ray photoelectron spectroscopy, respectively. The contact angle analysis shows that after purification the polar part of the surface energy increased to 17.5 from 12 m Jm À2 . While the surface area increased to an order of magnitude, i.e., 11.6 to 110.5 m 2 g À1 . The modification of purified silica particles with bis(3-triethoxysilylpropyl) tetrasulfane reveal the functionalization of hydroxyl to -Si-O-R groups as detected at 1560 cm À1 . As a consequence, the modified silica based S-SBR/BR composite resulted in improved mechanical properties due to enhanced silica-rubber interaction.

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“…The targeting vector (Fig. 2A) used for the creation of the mutant mouse line was produced by modifying pNTK [Mortensen, 2008], which was generously provided along with its nucleotide sequence by Dr. Richard M. Mortensen (University of Michigan, Ann Arbor). Two contiguous Apa L1 restriction fragments containing the WT C57BL6 Creb1 promoter and exon 1 (Insert 1, position −4575 to 1716, 6.3 kb), and a portion of the 17.8 kb intron 1 (Insert 2, position 1717 to 6938, 5.2 kb), were identified within BAC clone RP24‐528I8 using RestrictionMapper software (http://www.restrictionmapper.org/) and purified by preparative agarose gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

MBG/PLGA composite microspheres with prolonged drug release

Wang

Zhang

et al. 2008

J Biomed Mater Res

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Mesoporous bioactive glass (MBG) and composite microspheres with MBG particles embedded in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) matrix have been prepared and used to load gentamicin (GS). The in vitro drug release experiments from both MBG and composite microspheres were conducted in distilled water and phosphate buffered saline (PBS) solution at 37 degrees C for more than 30 days. In both water and PBS, GS release from the MBG was very fast with about 60 wt % of the loaded drug released in the first 24 h, and more than 80 wt % released in two days. MBG/PLGA composite microspheres showed an initial release of about 33 wt % in the first day, and 48 wt % in 2 days, and a subsequent sustained release lasting for more than 4 weeks in PBS. MBG/PLGA composite microspheres may be used as an alternative drug release system, especially as a bone void filler for bone repair due to their combined advantages of sustained release of antibiotics and apatite-forming ability.

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Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)

Cited by 7 publications

References 21 publications

MGST1, a GSH transferase/peroxidase essential for development and hematopoietic stem cell differentiation

MGST1, a GSH transferase/peroxidase essential for development and hematopoietic stem cell differentiation

Purification, surface modification of coal ash silica and its potential application in rubber composites

MBG/PLGA composite microspheres with prolonged drug release

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