https://wiki.fkkt.uni-lj.si/index.php?title=In%C5%BEenirstvo_spreminjanja_plazmatk_s_popravljanjem_primarnih_%C4%8Dlove%C5%A1kih_celic_B_na_osnovi_homologije&feed=atom&action=historyInženirstvo spreminjanja plazmatk s popravljanjem primarnih človeških celic B na osnovi homologije - Revision history2024-03-29T06:08:43ZRevision history for this page on the wikiMediaWiki 1.39.3https://wiki.fkkt.uni-lj.si/index.php?title=In%C5%BEenirstvo_spreminjanja_plazmatk_s_popravljanjem_primarnih_%C4%8Dlove%C5%A1kih_celic_B_na_osnovi_homologije&diff=15886&oldid=prevNives Ražnjević: /* Optimisation procedure */2019-05-23T14:39:32Z<p><span dir="auto"><span class="autocomment">Optimisation procedure</span></span></p>
<table style="background-color: #fff; color: #202122;" data-mw="interface">
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<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 14:39, 23 May 2019</td>
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<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>=Optimisation procedure=</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>=Optimisation procedure=</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>Prior to testing efficacy of B cells genome editing, respective optimisation of the experimental conditions had to be made. The latter has consisted of four crucial steps, namely: </div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>Prior to testing efficacy of B cells genome editing, respective optimisation of the experimental conditions had to be made. The latter has consisted of four crucial steps, namely: </div></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div># '''<del style="font-weight: bold; text-decoration: none;">Optimization </del>of cell expansion conditions''' – by the cultivation of B cells with the “B cell activation cocktail” approximately 36-fold expansion was achieved</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div># '''<ins style="font-weight: bold; text-decoration: none;">Optimisation </ins>of cell expansion conditions''' – by the cultivation of B cells with the “B cell activation cocktail” approximately 36-fold expansion was achieved</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># '''Screening the successfulness of construct insertion''' – two specific genome regions were selected for editing with RNP complexes containing respective guidance RNAs. Site-specific insertion was confirmed by Illumina sequencing.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># '''Screening the successfulness of construct insertion''' – two specific genome regions were selected for editing with RNP complexes containing respective guidance RNAs. Site-specific insertion was confirmed by Illumina sequencing.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># '''Screening the influence of genome editing region on the primary naïve human B cell differentiation''' – altogether, 5 genomic regions were selected: four of them are important for B cell differentiation (''IRF4, PRDM1, PAX5, BACH2''). The fifth region, ''CCR5'', has no significant influence on B cell differentiation. By disruption of the listed regions, their function was confirmed and thus appropriate region for insertion of factor IX sequence could be selected. Cell differentiation level was measured via flow cytometry. </div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># '''Screening the influence of genome editing region on the primary naïve human B cell differentiation''' – altogether, 5 genomic regions were selected: four of them are important for B cell differentiation (''IRF4, PRDM1, PAX5, BACH2''). The fifth region, ''CCR5'', has no significant influence on B cell differentiation. By disruption of the listed regions, their function was confirmed and thus appropriate region for insertion of factor IX sequence could be selected. Cell differentiation level was measured via flow cytometry. </div></td></tr>
</table>Nives Ražnjevićhttps://wiki.fkkt.uni-lj.si/index.php?title=In%C5%BEenirstvo_spreminjanja_plazmatk_s_popravljanjem_primarnih_%C4%8Dlove%C5%A1kih_celic_B_na_osnovi_homologije&diff=15884&oldid=prevNives Ražnjević: /* Conclusion */2019-05-23T14:29:22Z<p><span dir="auto"><span class="autocomment">Conclusion</span></span></p>
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<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 14:29, 23 May 2019</td>
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<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>=Conclusion=</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>=Conclusion=</div></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>In this research, conducted by Hung ''et al''., B cells were successfully used as a “cell factory” for production of active factor IX. Since the method of editing of B cells’ genome was CRISPR/Cas9 editing, the significance of the research lies in the opening opportunity for further usage the respective method to edit B cells’ genome for production of some other protein whose deficiency is causing anomalies. Also, optimisation conditions for the successful experiment were determined which sets up the appropriate ground for further research in this field such as studying mutations which may cause autoimmune diseases.</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>In this research, conducted by Hung ''et al''., B cells were successfully used as a “cell factory” for production of active factor IX. Since the method of editing of B cells’ genome was CRISPR/Cas9 editing, the significance of the research lies in the opening opportunity for further usage <ins style="font-weight: bold; text-decoration: none;">of </ins>the respective method to edit B cells’ genome for production of some other protein whose deficiency is causing anomalies. Also, optimisation conditions for the successful experiment were determined which sets up the appropriate ground for further research in this field such as studying mutations which may cause autoimmune diseases.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>=References=</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>=References=</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div><references/></div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div><references/></div></td></tr>
</table>Nives Ražnjevićhttps://wiki.fkkt.uni-lj.si/index.php?title=In%C5%BEenirstvo_spreminjanja_plazmatk_s_popravljanjem_primarnih_%C4%8Dlove%C5%A1kih_celic_B_na_osnovi_homologije&diff=15883&oldid=prevNives Ražnjević: /* Optimisation procedure */2019-05-23T14:26:58Z<p><span dir="auto"><span class="autocomment">Optimisation procedure</span></span></p>
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<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 14:26, 23 May 2019</td>
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<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># '''Screening the successfulness of construct insertion''' – two specific genome regions were selected for editing with RNP complexes containing respective guidance RNAs. Site-specific insertion was confirmed by Illumina sequencing.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># '''Screening the successfulness of construct insertion''' – two specific genome regions were selected for editing with RNP complexes containing respective guidance RNAs. Site-specific insertion was confirmed by Illumina sequencing.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># '''Screening the influence of genome editing region on the primary naïve human B cell differentiation''' – altogether, 5 genomic regions were selected: four of them are important for B cell differentiation (''IRF4, PRDM1, PAX5, BACH2''). The fifth region, ''CCR5'', has no significant influence on B cell differentiation. By disruption of the listed regions, their function was confirmed and thus appropriate region for insertion of factor IX sequence could be selected. Cell differentiation level was measured via flow cytometry. </div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># '''Screening the influence of genome editing region on the primary naïve human B cell differentiation''' – altogether, 5 genomic regions were selected: four of them are important for B cell differentiation (''IRF4, PRDM1, PAX5, BACH2''). The fifth region, ''CCR5'', has no significant influence on B cell differentiation. By disruption of the listed regions, their function was confirmed and thus appropriate region for insertion of factor IX sequence could be selected. Cell differentiation level was measured via flow cytometry. </div></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div># '''Selection of adequate template donor vector''' – scAAV was designed with the inserted sequence for GFP driven by MND promoter. GFP expression was measured by flow cytometry<del style="font-weight: bold; text-decoration: none;">, meanwhile</del>, minimal loss of cell viability was observed. B cells transduced with AAV serotype 6 showed the highest mean fluorescence.<ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref></div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div># '''Selection of adequate template donor vector''' – scAAV was designed with the inserted sequence for GFP driven by MND promoter. GFP expression was measured by flow cytometry<ins style="font-weight: bold; text-decoration: none;">. Meanwhile</ins>, minimal loss of cell viability was observed. B cells transduced with AAV serotype 6 showed the highest mean fluorescence.<ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref></div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>=Testing the efficacy of genome editing=</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>=Testing the efficacy of genome editing=</div></td></tr>
</table>Nives Ražnjevićhttps://wiki.fkkt.uni-lj.si/index.php?title=In%C5%BEenirstvo_spreminjanja_plazmatk_s_popravljanjem_primarnih_%C4%8Dlove%C5%A1kih_celic_B_na_osnovi_homologije&diff=15882&oldid=prevNives Ražnjević: /* Optimisation procedure */2019-05-23T14:26:05Z<p><span dir="auto"><span class="autocomment">Optimisation procedure</span></span></p>
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<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># '''Screening the successfulness of construct insertion''' – two specific genome regions were selected for editing with RNP complexes containing respective guidance RNAs. Site-specific insertion was confirmed by Illumina sequencing.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># '''Screening the successfulness of construct insertion''' – two specific genome regions were selected for editing with RNP complexes containing respective guidance RNAs. Site-specific insertion was confirmed by Illumina sequencing.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># '''Screening the influence of genome editing region on the primary naïve human B cell differentiation''' – altogether, 5 genomic regions were selected: four of them are important for B cell differentiation (''IRF4, PRDM1, PAX5, BACH2''). The fifth region, ''CCR5'', has no significant influence on B cell differentiation. By disruption of the listed regions, their function was confirmed and thus appropriate region for insertion of factor IX sequence could be selected. Cell differentiation level was measured via flow cytometry. </div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># '''Screening the influence of genome editing region on the primary naïve human B cell differentiation''' – altogether, 5 genomic regions were selected: four of them are important for B cell differentiation (''IRF4, PRDM1, PAX5, BACH2''). The fifth region, ''CCR5'', has no significant influence on B cell differentiation. By disruption of the listed regions, their function was confirmed and thus appropriate region for insertion of factor IX sequence could be selected. Cell differentiation level was measured via flow cytometry. </div></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div># '''Selection of adequate template donor vector''' – scAAV was designed with inserted GFP <del style="font-weight: bold; text-decoration: none;">sequence </del>driven by MND promoter. GFP expression was measured by flow cytometry, meanwhile, minimal loss of cell viability was observed. B cells transduced with AAV serotype 6 showed the highest mean fluorescence.<ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref> </div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div># '''Selection of adequate template donor vector''' – scAAV was designed with <ins style="font-weight: bold; text-decoration: none;">the </ins>inserted <ins style="font-weight: bold; text-decoration: none;">sequence for </ins>GFP driven by MND promoter. GFP expression was measured by flow cytometry, meanwhile, minimal loss of cell viability was observed. B cells transduced with AAV serotype 6 showed the highest mean fluorescence.<ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref></div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>=Testing the efficacy of genome editing=</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>=Testing the efficacy of genome editing=</div></td></tr>
</table>Nives Ražnjevićhttps://wiki.fkkt.uni-lj.si/index.php?title=In%C5%BEenirstvo_spreminjanja_plazmatk_s_popravljanjem_primarnih_%C4%8Dlove%C5%A1kih_celic_B_na_osnovi_homologije&diff=15881&oldid=prevNives Ražnjević at 14:23, 23 May 20192019-05-23T14:23:58Z<p></p>
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<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>[https://www.hemophilia.org/Bleeding-Disorders/Types-of-Bleeding-Disorders/Hemophilia-B Haemophilia B] is a disease caused by a deficiency in the secretion of coagulation factor IX (FIX) which results in the impaired blood coagulation cascade<ref name="ena">Nathwani ''et al''. (2011). Adenovirusassociated virus vector-mediated gene transfer in hemophilia B. N. ''Engl. J. Med.'' 365, 2357–2365</ref>. Thus, the disease is manifested by blood dotting defects. Considering the plasma cells’ ability to produce ''de novo'' protein, it opens up an opportunity for the production of deficient protein and thus the potential cure for protein-deficiency diseases. This review article will focus on the methods used to investigate the ability of activated B cells differentiated from primary naive human B cells to produce functioning factor IX by [https://www.addgene.org/crispr/guide/CRISPR/Cas9 CRISPR/Cas9] genome editing tool and [https://blog.addgene.org/crispr-101-homology-directed-repair homology-directed repair (HDR)] <ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref>. </div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>[https://www.hemophilia.org/Bleeding-Disorders/Types-of-Bleeding-Disorders/Hemophilia-B Haemophilia B] is a disease caused by a deficiency in the secretion of coagulation factor IX (FIX) which results in the impaired blood coagulation cascade<ref name="ena">Nathwani ''et al''. (2011). Adenovirusassociated virus vector-mediated gene transfer in hemophilia B. N. ''Engl. J. Med.'' 365, 2357–2365</ref>. Thus, the disease is manifested by blood dotting defects. Considering the plasma cells’ ability to produce ''de novo'' protein, it opens up an opportunity for the production of deficient protein and thus the potential cure for protein-deficiency diseases. This review article will focus on the methods used to investigate the ability of activated B cells differentiated from primary naive human B cells to produce functioning factor IX <ins style="font-weight: bold; text-decoration: none;">edited </ins>by [https://www.addgene.org/crispr/guide/CRISPR/Cas9 CRISPR/Cas9] genome editing tool and [https://blog.addgene.org/crispr-101-homology-directed-repair homology-directed repair (HDR)] <ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref>. </div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>=Optimisation procedure=</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>=Optimisation procedure=</div></td></tr>
</table>Nives Ražnjevićhttps://wiki.fkkt.uni-lj.si/index.php?title=In%C5%BEenirstvo_spreminjanja_plazmatk_s_popravljanjem_primarnih_%C4%8Dlove%C5%A1kih_celic_B_na_osnovi_homologije&diff=15880&oldid=prevNives Ražnjević at 14:22, 23 May 20192019-05-23T14:22:00Z<p></p>
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<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 14:22, 23 May 2019</td>
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<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>[https://www.hemophilia.org/Bleeding-Disorders/Types-of-Bleeding-Disorders/Hemophilia-B Haemophilia B] is a disease caused by a deficiency in the secretion of coagulation factor IX (FIX) which results in the impaired blood coagulation cascade<ref name="ena">Nathwani ''et al''. (2011). Adenovirusassociated virus vector-mediated gene transfer in hemophilia B. N. ''Engl. J. Med.'' 365, 2357–2365</ref>. Thus, the disease is manifested by blood dotting defects. Considering the plasma cells’ ability to produce ''de novo'' protein, it opens up an opportunity for the production of deficient protein <del style="font-weight: bold; text-decoration: none;">(in the terms of the disease) </del>and thus the potential cure for protein-deficiency diseases. This review article will focus on the methods used to investigate the ability of activated B cells differentiated from primary naive human B cells to produce functioning factor IX by [https://www.addgene.org/crispr/guide/CRISPR/Cas9 CRISPR/Cas9] genome editing tool and [https://blog.addgene.org/crispr-101-homology-directed-repair homology-directed repair (HDR)] <ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref>. </div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>[https://www.hemophilia.org/Bleeding-Disorders/Types-of-Bleeding-Disorders/Hemophilia-B Haemophilia B] is a disease caused by a deficiency in the secretion of coagulation factor IX (FIX) which results in the impaired blood coagulation cascade<ref name="ena">Nathwani ''et al''. (2011). Adenovirusassociated virus vector-mediated gene transfer in hemophilia B. N. ''Engl. J. Med.'' 365, 2357–2365</ref>. Thus, the disease is manifested by blood dotting defects. Considering the plasma cells’ ability to produce ''de novo'' protein, it opens up an opportunity for the production of deficient protein and thus the potential cure for protein-deficiency diseases. This review article will focus on the methods used to investigate the ability of activated B cells differentiated from primary naive human B cells to produce functioning factor IX by [https://www.addgene.org/crispr/guide/CRISPR/Cas9 CRISPR/Cas9] genome editing tool and [https://blog.addgene.org/crispr-101-homology-directed-repair homology-directed repair (HDR)] <ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref>. </div></td></tr>
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<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>=Optimisation procedure=</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>=Optimisation procedure=</div></td></tr>
</table>Nives Ražnjevićhttps://wiki.fkkt.uni-lj.si/index.php?title=In%C5%BEenirstvo_spreminjanja_plazmatk_s_popravljanjem_primarnih_%C4%8Dlove%C5%A1kih_celic_B_na_osnovi_homologije&diff=15879&oldid=prevNives Ražnjević at 14:20, 23 May 20192019-05-23T14:20:58Z<p></p>
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<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 14:20, 23 May 2019</td>
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<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>[https://www.hemophilia.org/Bleeding-Disorders/Types-of-Bleeding-Disorders/Hemophilia-B Haemophilia B] is a disease caused by a deficiency in the secretion of coagulation factor IX (FIX) which results in the impaired blood coagulation cascade<ref name="ena">Nathwani ''et al''. (2011). Adenovirusassociated virus vector-mediated gene transfer in hemophilia B. N. ''Engl. J. Med.'' 365, 2357–2365</ref>. Thus, the disease is manifested by blood dotting defects. Considering the plasma cells’ ability to produce de novo protein, it opens up an opportunity for the production of deficient protein (in the terms of the disease) and thus the potential cure for protein-deficiency diseases. This review article will focus on the methods used to investigate the ability of activated B cells differentiated from primary naive human B cells to produce functioning factor IX by [https://www.addgene.org/crispr/guide/CRISPR/Cas9 CRISPR/Cas9] genome editing tool and [https://blog.addgene.org/crispr-101-homology-directed-repair homology-directed repair (HDR)] <ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref>. </div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>[https://www.hemophilia.org/Bleeding-Disorders/Types-of-Bleeding-Disorders/Hemophilia-B Haemophilia B] is a disease caused by a deficiency in the secretion of coagulation factor IX (FIX) which results in the impaired blood coagulation cascade<ref name="ena">Nathwani ''et al''. (2011). Adenovirusassociated virus vector-mediated gene transfer in hemophilia B. N. ''Engl. J. Med.'' 365, 2357–2365</ref>. Thus, the disease is manifested by blood dotting defects. Considering the plasma cells’ ability to produce <ins style="font-weight: bold; text-decoration: none;">''</ins>de novo<ins style="font-weight: bold; text-decoration: none;">'' </ins>protein, it opens up an opportunity for the production of deficient protein (in the terms of the disease) and thus the potential cure for protein-deficiency diseases. This review article will focus on the methods used to investigate the ability of activated B cells differentiated from primary naive human B cells to produce functioning factor IX by [https://www.addgene.org/crispr/guide/CRISPR/Cas9 CRISPR/Cas9] genome editing tool and [https://blog.addgene.org/crispr-101-homology-directed-repair homology-directed repair (HDR)] <ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref>. </div></td></tr>
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</table>Nives Ražnjevićhttps://wiki.fkkt.uni-lj.si/index.php?title=In%C5%BEenirstvo_spreminjanja_plazmatk_s_popravljanjem_primarnih_%C4%8Dlove%C5%A1kih_celic_B_na_osnovi_homologije&diff=15877&oldid=prevNives Ražnjević: New page: [https://www.hemophilia.org/Bleeding-Disorders/Types-of-Bleeding-Disorders/Hemophilia-B Haemophilia B] is a disease caused by a deficiency in the secretion of coagulation factor IX (FIX) w...2019-05-23T14:17:43Z<p>New page: [https://www.hemophilia.org/Bleeding-Disorders/Types-of-Bleeding-Disorders/Hemophilia-B Haemophilia B] is a disease caused by a deficiency in the secretion of coagulation factor IX (FIX) w...</p>
<p><b>New page</b></p><div>[https://www.hemophilia.org/Bleeding-Disorders/Types-of-Bleeding-Disorders/Hemophilia-B Haemophilia B] is a disease caused by a deficiency in the secretion of coagulation factor IX (FIX) which results in the impaired blood coagulation cascade<ref name="ena">Nathwani ''et al''. (2011). Adenovirusassociated virus vector-mediated gene transfer in hemophilia B. N. ''Engl. J. Med.'' 365, 2357–2365</ref>. Thus, the disease is manifested by blood dotting defects. Considering the plasma cells’ ability to produce de novo protein, it opens up an opportunity for the production of deficient protein (in the terms of the disease) and thus the potential cure for protein-deficiency diseases. This review article will focus on the methods used to investigate the ability of activated B cells differentiated from primary naive human B cells to produce functioning factor IX by [https://www.addgene.org/crispr/guide/CRISPR/Cas9 CRISPR/Cas9] genome editing tool and [https://blog.addgene.org/crispr-101-homology-directed-repair homology-directed repair (HDR)] <ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref>. <br />
<br />
=Optimisation procedure=<br />
Prior to testing efficacy of B cells genome editing, respective optimisation of the experimental conditions had to be made. The latter has consisted of four crucial steps, namely: <br />
# '''Optimization of cell expansion conditions''' – by the cultivation of B cells with the “B cell activation cocktail” approximately 36-fold expansion was achieved<br />
# '''Screening the successfulness of construct insertion''' – two specific genome regions were selected for editing with RNP complexes containing respective guidance RNAs. Site-specific insertion was confirmed by Illumina sequencing.<br />
# '''Screening the influence of genome editing region on the primary naïve human B cell differentiation''' – altogether, 5 genomic regions were selected: four of them are important for B cell differentiation (''IRF4, PRDM1, PAX5, BACH2''). The fifth region, ''CCR5'', has no significant influence on B cell differentiation. By disruption of the listed regions, their function was confirmed and thus appropriate region for insertion of factor IX sequence could be selected. Cell differentiation level was measured via flow cytometry. <br />
# '''Selection of adequate template donor vector''' – scAAV was designed with inserted GFP sequence driven by MND promoter. GFP expression was measured by flow cytometry, meanwhile, minimal loss of cell viability was observed. B cells transduced with AAV serotype 6 showed the highest mean fluorescence.<ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref> <br />
<br />
=Testing the efficacy of genome editing=<br />
The genome of primary human B cells was edited in terms of insertion of two genes – one for factor IX (whose production was the main goal of the research) and the other for BAFF (B cell activating factor)<ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref>. <br />
<br />
==Insertion and expression of factor IX==<br />
The region used for genome editing was ''CCR5'' region of the B cells’ genome for which it was shown (in prior experimental conditions testing) that it has no significant effect on B cell differentiation if edited. Two parallel experiments were conducted, the one containing only Cas9 RNP with gRNA for the respective region and AAV with donor template for insertion of FIX and the other, where under the same conditions of FIX insertion, ''PAX5'' region was inhibited to induce a higher rate of B cell differentiation. However, no significant difference in differentiation was shown. <br />
The insertion of factor IX nucleotide sequence was successful, as well as the expression of the respective protein in comparison to control. The presence of produced factor IX was determined by ELISA method. The activity of produced factor IX was tested afterwards with chromogenic assay and high-specific-activity has been shown in the vitamin K supplemented culture<ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref>. <br />
<br />
==Insertion and expression of BAFF==<br />
For BAFF it is known that it provides activation and survival of B cells. The idea is that naïve primary B cells would be doubly edited, which means that while expressing functioning factor IX, it would at the same time produce enough BAFF for its own sustainment by autocrine secretion. The expression of BAFF was determined by ELISA method<ref name="dve">Hung ''et al''. (2018). Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. ''Mol. Ther.'' 26(2), 456-467.</ref>. <br />
<br />
=Conclusion=<br />
In this research, conducted by Hung ''et al''., B cells were successfully used as a “cell factory” for production of active factor IX. Since the method of editing of B cells’ genome was CRISPR/Cas9 editing, the significance of the research lies in the opening opportunity for further usage the respective method to edit B cells’ genome for production of some other protein whose deficiency is causing anomalies. Also, optimisation conditions for the successful experiment were determined which sets up the appropriate ground for further research in this field such as studying mutations which may cause autoimmune diseases.<br />
<br />
=References=<br />
<references/></div>Nives Ražnjević