Development of a Transparent Transgenic Zebrafish Cellular Phenotype <i>Tg</i>(<i>6xNF-kB:EGFP); Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>) to Study NF-kB Activity
NF-κB signaling has broad effects on cell survival, tissue growth, and proliferation activities. It controls many genes that are involved in inflammation and thus is a key player in many inflammatory diseases. The elevation of NF-κB activators is associated with elevated mortality, especially in can...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-07-01
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| Series: | Biomedicines |
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| Online Access: | https://www.mdpi.com/2227-9059/11/7/1985 |
| _version_ | 1797590190646099968 |
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| author | Surendra K. Rajpurohit Logan Ouellette Suvarsha Sura Chelsea Appiah Annabelle O’Keefe Katherine McCarthy Umasai Kandepu May Ye Mon Kirk Kimmerling Vishal Arora Bal L. Lokeshwar |
| author_facet | Surendra K. Rajpurohit Logan Ouellette Suvarsha Sura Chelsea Appiah Annabelle O’Keefe Katherine McCarthy Umasai Kandepu May Ye Mon Kirk Kimmerling Vishal Arora Bal L. Lokeshwar |
| author_sort | Surendra K. Rajpurohit |
| collection | DOAJ |
| description | NF-κB signaling has broad effects on cell survival, tissue growth, and proliferation activities. It controls many genes that are involved in inflammation and thus is a key player in many inflammatory diseases. The elevation of NF-κB activators is associated with elevated mortality, especially in cancer and cardiovascular diseases. The zebrafish has emerged as an important model for whole-organism in vivo modeling in translational research. In vertebrates, in-vivo spatial resolution is limited due to normal opacification of skin and subdermal structure. For in vivo imaging, skin transparency by blocking the pigmentation via chemical inhibition is required and the maintenance of this transparency is vital. The <i>Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>) mutant of zebrafish maintains this transparency throughout its life and serves as an ideal combination of sensitivity and resolution for in vivo stem cell analyses and imaging. We developed an NF-kB:GFP/Casper transparent transgenic zebrafish cellular phenotype to study inflammatory processes in vivo. We outline the experimental setup to generate a transparent transgenic <i>NF-kB</i>/<i>Casper</i> strain of zebrafish through the cross-breeding of <i>Casper</i> and NF-kB transgenic adult fish and have generated F01 in the form of heterozygous progeny. The transgenic F01 progeny was further inbred to generate heterozygous progenies from F1 to F4 generations. Furthermore, it continued to successfully develop the homozygous strain <i>Tg</i>(<i>6xNF-kB:EGFP); Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>) in the F05 generation. This novel strain of F05 generation showed 100% homozygosity in the transgenic transparent progeny of <i>Tg</i>(<i>6xNF-kB:EGFP)</i>; <i>Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>). The strain has been confirmed by generating the F06 generation of homozygous progeny and again verified and validated for its homogeneity in the F07 generation. The newly developed novel transparent transgenic strain of the NF-kB reporter line has been coined as “<i>Tg</i>(<i>6xNF-kB:EGFP)</i>; <i>Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>)<i>gmc1</i>”. We have established a newly generated phenotype of transparent transgenic zebrafish for time-lapse in vivo confocal microscopy to study the cellular phenotype and pathologies at the cellular level over time. This will allow for quantifying the changes in the NF-kB functional activities over time and allow the comparison of control and cardiac-oncology experimental therapeutics. We validated the newly developed <i>Tg</i>(<i>6xNF-kB:EGFP)</i>; <i>Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>)<i>gmc1</i> homozygous strain of zebrafish by studying the inflammatory response to bacterial lipopolysaccharide (LPS) exposure, tolerance, and the inhibitory role of a potential novel drug candidate against LPS-induced inflammation. The results establish the unique application of newly developed strains by identifying hit and lead drug candidates for experimental therapeutics. |
| first_indexed | 2024-03-11T01:17:00Z |
| format | Article |
| id | doaj.art-c6ac4d19ace6431a91bc5837a0d0e6cf |
| institution | Directory Open Access Journal |
| issn | 2227-9059 |
| language | English |
| last_indexed | 2024-03-11T01:17:00Z |
| publishDate | 2023-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomedicines |
| spelling | doaj.art-c6ac4d19ace6431a91bc5837a0d0e6cf2023-11-18T18:27:49ZengMDPI AGBiomedicines2227-90592023-07-01117198510.3390/biomedicines11071985Development of a Transparent Transgenic Zebrafish Cellular Phenotype <i>Tg</i>(<i>6xNF-kB:EGFP); Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>) to Study NF-kB ActivitySurendra K. Rajpurohit0Logan Ouellette1Suvarsha Sura2Chelsea Appiah3Annabelle O’Keefe4Katherine McCarthy5Umasai Kandepu6May Ye Mon7Kirk Kimmerling8Vishal Arora9Bal L. Lokeshwar10Georgia Cancer Center, Medical College of Georgia, Augusta University, Augusta, GA 30912, USAGeorgia Cancer Center, Medical College of Georgia, Augusta University, Augusta, GA 30912, USAGeorgia Cancer Center, Medical College of Georgia, Augusta University, Augusta, GA 30912, USAGeorgia Cancer Center, Medical College of Georgia, Augusta University, Augusta, GA 30912, USAGeorgia Cancer Center, Medical College of Georgia, Augusta University, Augusta, GA 30912, USAGeorgia Cancer Center, Medical College of Georgia, Augusta University, Augusta, GA 30912, USAGeorgia Cancer Center, Medical College of Georgia, Augusta University, Augusta, GA 30912, USAGeorgia Cancer Center, Medical College of Georgia, Augusta University, Augusta, GA 30912, USAKHG FiteBac Technology, Marietta, GA 30064, USADivision of Cardiology, Department of Medicine, Medical College of Georgia, Augusta University, Augusta, GA 30912, USAGeorgia Cancer Center, Medical College of Georgia, Augusta University, Augusta, GA 30912, USANF-κB signaling has broad effects on cell survival, tissue growth, and proliferation activities. It controls many genes that are involved in inflammation and thus is a key player in many inflammatory diseases. The elevation of NF-κB activators is associated with elevated mortality, especially in cancer and cardiovascular diseases. The zebrafish has emerged as an important model for whole-organism in vivo modeling in translational research. In vertebrates, in-vivo spatial resolution is limited due to normal opacification of skin and subdermal structure. For in vivo imaging, skin transparency by blocking the pigmentation via chemical inhibition is required and the maintenance of this transparency is vital. The <i>Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>) mutant of zebrafish maintains this transparency throughout its life and serves as an ideal combination of sensitivity and resolution for in vivo stem cell analyses and imaging. We developed an NF-kB:GFP/Casper transparent transgenic zebrafish cellular phenotype to study inflammatory processes in vivo. We outline the experimental setup to generate a transparent transgenic <i>NF-kB</i>/<i>Casper</i> strain of zebrafish through the cross-breeding of <i>Casper</i> and NF-kB transgenic adult fish and have generated F01 in the form of heterozygous progeny. The transgenic F01 progeny was further inbred to generate heterozygous progenies from F1 to F4 generations. Furthermore, it continued to successfully develop the homozygous strain <i>Tg</i>(<i>6xNF-kB:EGFP); Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>) in the F05 generation. This novel strain of F05 generation showed 100% homozygosity in the transgenic transparent progeny of <i>Tg</i>(<i>6xNF-kB:EGFP)</i>; <i>Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>). The strain has been confirmed by generating the F06 generation of homozygous progeny and again verified and validated for its homogeneity in the F07 generation. The newly developed novel transparent transgenic strain of the NF-kB reporter line has been coined as “<i>Tg</i>(<i>6xNF-kB:EGFP)</i>; <i>Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>)<i>gmc1</i>”. We have established a newly generated phenotype of transparent transgenic zebrafish for time-lapse in vivo confocal microscopy to study the cellular phenotype and pathologies at the cellular level over time. This will allow for quantifying the changes in the NF-kB functional activities over time and allow the comparison of control and cardiac-oncology experimental therapeutics. We validated the newly developed <i>Tg</i>(<i>6xNF-kB:EGFP)</i>; <i>Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>)<i>gmc1</i> homozygous strain of zebrafish by studying the inflammatory response to bacterial lipopolysaccharide (LPS) exposure, tolerance, and the inhibitory role of a potential novel drug candidate against LPS-induced inflammation. The results establish the unique application of newly developed strains by identifying hit and lead drug candidates for experimental therapeutics.https://www.mdpi.com/2227-9059/11/7/1985zebrafishtransgenictransparentcasperNF-kBinflammation |
| spellingShingle | Surendra K. Rajpurohit Logan Ouellette Suvarsha Sura Chelsea Appiah Annabelle O’Keefe Katherine McCarthy Umasai Kandepu May Ye Mon Kirk Kimmerling Vishal Arora Bal L. Lokeshwar Development of a Transparent Transgenic Zebrafish Cellular Phenotype <i>Tg</i>(<i>6xNF-kB:EGFP); Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>) to Study NF-kB Activity Biomedicines zebrafish transgenic transparent casper NF-kB inflammation |
| title | Development of a Transparent Transgenic Zebrafish Cellular Phenotype <i>Tg</i>(<i>6xNF-kB:EGFP); Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>) to Study NF-kB Activity |
| title_full | Development of a Transparent Transgenic Zebrafish Cellular Phenotype <i>Tg</i>(<i>6xNF-kB:EGFP); Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>) to Study NF-kB Activity |
| title_fullStr | Development of a Transparent Transgenic Zebrafish Cellular Phenotype <i>Tg</i>(<i>6xNF-kB:EGFP); Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>) to Study NF-kB Activity |
| title_full_unstemmed | Development of a Transparent Transgenic Zebrafish Cellular Phenotype <i>Tg</i>(<i>6xNF-kB:EGFP); Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>) to Study NF-kB Activity |
| title_short | Development of a Transparent Transgenic Zebrafish Cellular Phenotype <i>Tg</i>(<i>6xNF-kB:EGFP); Casper</i>(<i>roy</i><sup>−/−</sup>, <i>nacre</i><sup>−/−</sup>) to Study NF-kB Activity |
| title_sort | development of a transparent transgenic zebrafish cellular phenotype i tg i i 6xnf kb egfp casper i i roy i sup sup i nacre i sup sup to study nf kb activity |
| topic | zebrafish transgenic transparent casper NF-kB inflammation |
| url | https://www.mdpi.com/2227-9059/11/7/1985 |
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