Given the limited data describing the phenotype for type 2 interleukin -1 receptor (Il1r2-/-) in mouse strains and based on the decoy role of the gene product (IL-1R2), we hypothesized that IL-1R2 may have a unique but similar inhibitory role to IL-1R antagonist (IL-1Ra) in vivo. Despite the anti-inflammatory function of IL-1R2, its role in disease in vivo remains unclear. Therefore, we designed and implemented a knock-out construct template for a mouse embryonic stem (ES) cell line that can be used to easily make knockout mice. Bacterial artificial chromosome (BAC) clone of Il1r2 from mouse strain AB2.2 and ES cells from same strain were obtained and a long chain PCR was performed to isolate homologous arms containing homologous segments (8kb and 2kb). Short segments were cloned out for use as probe sequences and a construct with deletion in exon 3 was made so it can be removed in vivo. The NeoR in cloning vector was then flanked with loxP elements. The two homologous arms were successfully amplified with a truncation in exon 3 of the gene and the wild type cloning plasmid (p1049) was serially modified with loxP elements. One of the successfully transformed plasmid DNA was used as the starting material for the ligation of the subsequent loxP-PacI linker. This plasmid (p1049XL) was amplified in E. coli DH5α cells and DNA extracted. The loxP-PacI linker was successfully ligated into the plasmid and transformed clones screened with MspI and compared to the virtual digest of the theoretical plasmid containing the insert and subsequently sequenced. This study has provided the basic ingredients for making an Il1r2-deficient mouse in order to adequately characterize the phenotype. By assembling the complete knock out construct from templates already provided in this study for the knock out in embryonic stem cells, Il1r2-deficient mice could be made.
| Published in | American Journal of Biomedical and Life Sciences (Volume 7, Issue 1) |
| DOI | 10.11648/j.ajbls.20190701.15 |
| Page(s) | 22-30 |
| Creative Commons |
This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited. |
| Copyright |
Copyright © The Author(s), 2019. Published by Science Publishing Group |
IL-1R2, Embryonic Stem Cell, BAC Clone, Long Chain PCR, Loxp Elements, Phenotype, Homologous Segments
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APA Style
Faith Owabhel Robert, Emmanuel Amabebe. (2019). Rational Design and Construction of a Knock-Out Vector for Targeting the Decoy Type 2 Interleukin -1 Receptor. American Journal of Biomedical and Life Sciences, 7(1), 22-30. https://doi.org/10.11648/j.ajbls.20190701.15
ACS Style
Faith Owabhel Robert; Emmanuel Amabebe. Rational Design and Construction of a Knock-Out Vector for Targeting the Decoy Type 2 Interleukin -1 Receptor. Am. J. Biomed. Life Sci. 2019, 7(1), 22-30. doi: 10.11648/j.ajbls.20190701.15
AMA Style
Faith Owabhel Robert, Emmanuel Amabebe. Rational Design and Construction of a Knock-Out Vector for Targeting the Decoy Type 2 Interleukin -1 Receptor. Am J Biomed Life Sci. 2019;7(1):22-30. doi: 10.11648/j.ajbls.20190701.15
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Download@article{10.11648/j.ajbls.20190701.15,
author = {Faith Owabhel Robert and Emmanuel Amabebe},
title = {Rational Design and Construction of a Knock-Out Vector for Targeting the Decoy Type 2 Interleukin -1 Receptor},
journal = {American Journal of Biomedical and Life Sciences},
volume = {7},
number = {1},
pages = {22-30},
doi = {10.11648/j.ajbls.20190701.15},
url = {https://doi.org/10.11648/j.ajbls.20190701.15},
eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ajbls.20190701.15},
abstract = {Given the limited data describing the phenotype for type 2 interleukin -1 receptor (Il1r2-/-) in mouse strains and based on the decoy role of the gene product (IL-1R2), we hypothesized that IL-1R2 may have a unique but similar inhibitory role to IL-1R antagonist (IL-1Ra) in vivo. Despite the anti-inflammatory function of IL-1R2, its role in disease in vivo remains unclear. Therefore, we designed and implemented a knock-out construct template for a mouse embryonic stem (ES) cell line that can be used to easily make knockout mice. Bacterial artificial chromosome (BAC) clone of Il1r2 from mouse strain AB2.2 and ES cells from same strain were obtained and a long chain PCR was performed to isolate homologous arms containing homologous segments (8kb and 2kb). Short segments were cloned out for use as probe sequences and a construct with deletion in exon 3 was made so it can be removed in vivo. The NeoR in cloning vector was then flanked with loxP elements. The two homologous arms were successfully amplified with a truncation in exon 3 of the gene and the wild type cloning plasmid (p1049) was serially modified with loxP elements. One of the successfully transformed plasmid DNA was used as the starting material for the ligation of the subsequent loxP-PacI linker. This plasmid (p1049XL) was amplified in E. coli DH5α cells and DNA extracted. The loxP-PacI linker was successfully ligated into the plasmid and transformed clones screened with MspI and compared to the virtual digest of the theoretical plasmid containing the insert and subsequently sequenced. This study has provided the basic ingredients for making an Il1r2-deficient mouse in order to adequately characterize the phenotype. By assembling the complete knock out construct from templates already provided in this study for the knock out in embryonic stem cells, Il1r2-deficient mice could be made.},
year = {2019}
}
TY - JOUR T1 - Rational Design and Construction of a Knock-Out Vector for Targeting the Decoy Type 2 Interleukin -1 Receptor AU - Faith Owabhel Robert AU - Emmanuel Amabebe Y1 - 2019/04/22 PY - 2019 N1 - https://doi.org/10.11648/j.ajbls.20190701.15 DO - 10.11648/j.ajbls.20190701.15 T2 - American Journal of Biomedical and Life Sciences JF - American Journal of Biomedical and Life Sciences JO - American Journal of Biomedical and Life Sciences SP - 22 EP - 30 PB - Science Publishing Group SN - 2330-880X UR - https://doi.org/10.11648/j.ajbls.20190701.15 AB - Given the limited data describing the phenotype for type 2 interleukin -1 receptor (Il1r2-/-) in mouse strains and based on the decoy role of the gene product (IL-1R2), we hypothesized that IL-1R2 may have a unique but similar inhibitory role to IL-1R antagonist (IL-1Ra) in vivo. Despite the anti-inflammatory function of IL-1R2, its role in disease in vivo remains unclear. Therefore, we designed and implemented a knock-out construct template for a mouse embryonic stem (ES) cell line that can be used to easily make knockout mice. Bacterial artificial chromosome (BAC) clone of Il1r2 from mouse strain AB2.2 and ES cells from same strain were obtained and a long chain PCR was performed to isolate homologous arms containing homologous segments (8kb and 2kb). Short segments were cloned out for use as probe sequences and a construct with deletion in exon 3 was made so it can be removed in vivo. The NeoR in cloning vector was then flanked with loxP elements. The two homologous arms were successfully amplified with a truncation in exon 3 of the gene and the wild type cloning plasmid (p1049) was serially modified with loxP elements. One of the successfully transformed plasmid DNA was used as the starting material for the ligation of the subsequent loxP-PacI linker. This plasmid (p1049XL) was amplified in E. coli DH5α cells and DNA extracted. The loxP-PacI linker was successfully ligated into the plasmid and transformed clones screened with MspI and compared to the virtual digest of the theoretical plasmid containing the insert and subsequently sequenced. This study has provided the basic ingredients for making an Il1r2-deficient mouse in order to adequately characterize the phenotype. By assembling the complete knock out construct from templates already provided in this study for the knock out in embryonic stem cells, Il1r2-deficient mice could be made. VL - 7 IS - 1 ER -
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