Name: genome engineering of primary human b cells using crispr/cas9
Uploaded: 2020-11-03T14:00:00
Description: Watch this Scientific Journal Video about Genome Engineering of Primary Human B Cells Using CRISPR/Cas9 at JoVE.com

Bu çalışma Çocuk Kanser araştırma fonu (CCRF) ve NIH R01 AI146009 tarafından B.S.M'a finanse edildi.

Sağlıklı donörlerden lökerez örnekleri yerel bir kan bankasından alındı. Burada açıklanan tüm deneyler Kurumsal İnceleme Kurulu (IRB) tarafından araştırmadan muaf olarak belirlenmiş ve Minnesota Üniversitesi Kurumsal Biyogüvenlik Komitesi (IBC) tarafından onaylanmıştır.
NOT: Tüm deneyler kan kaynaklı patojenler için evrensel önlem doğrultusunda, steril/aseptik teknikler ve uygun biyogüvenlik seviye-2 ekipmanları ile gerçek gerçekleştirildi.
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+CD40+ligand+stimulates+B+cell+proliferation+and+immunoglobulin+E+secretion.">Recombinant human CD40 ligand stimulates B cell proliferation and immunoglobulin E secretion.</a> Journal of Experimental Medicines. 176 (6), 1543-1550 (1992).</li><li>Fusil, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lentiviral+vector+allowing+physiologically+regulated+membrane-anchored+and+secreted+antibody+expression+depending+on+B-cell+maturation+status.">A lentiviral vector allowing physiologically regulated membrane-anchored and secreted antibody expression depending on B-cell maturation status.</a> Molecular Therapy. 23 (11), 1734-1747 (2015).</li><li>Luo, X. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+human+hematopoietic+stem/progenitor+cells+to+produce+a+broadly+neutralizing+anti-HIV+antibody+after+in+vitro+maturation+to+human+B+lymphocytes.">Engineering human hematopoietic stem/progenitor cells to produce a broadly neutralizing anti-HIV antibody after in vitro maturation to human B lymphocytes.</a> Blood. 113 (7), 1422-1431 (2008).</li><li>Mock, U., Thiele, R., Uhde, A., Fehse, B., Horn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+lentiviral+transduction+and+transgene+expression+in+primary+human+B+cells.">Efficient lentiviral transduction and transgene expression in primary human B cells.</a> Human Gene Therapy Methods. 23 (6), 408-415 (2012).</li><li>Heltemes-Harris, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleeping+Beauty+transposon+screen+identifies+signaling+modules+that+cooperate+with+STAT5+activation+to+induce+B-cell+acute+lymphoblastic+leukemia.">Sleeping Beauty transposon screen identifies signaling modules that cooperate with STAT5 activation to induce B-cell acute lymphoblastic leukemia.</a> Oncogene. 35 (26), 3454-3464 (2016).</li><li>Johnson, M. J., Laoharawee, K., Lahr, W. S., Webber, B. R., Moriarity, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+primary+human+B+cells+with+CRISPR/Cas9+targeted+nuclease.">Engineering of primary human B cells with CRISPR/Cas9 targeted nuclease.</a> Scientific Reports. 8, 12144 (2018).</li><li>Hung, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+protein-secreting+plasma+cells+by+homology-directed+repair+in+primary+human+B+cells.">Engineering protein-secreting plasma cells by homology-directed repair in primary human B cells.</a> Molecular Therapy. 26 (2), 456-467 (2018).</li><li>Cui, Y., Xu, J., Cheng, M., Liao, X., Peng, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+CRISPR/Cas9+sgRNA+design+tools.">Review of CRISPR/Cas9 sgRNA design tools.</a> Interdisciplinary Sciences. 10 (2), 455-465 (2018).</li><li>Rees, H. A., Liu, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Base+editing:+precision+chemistry+on+the+genome+and+transcriptome+of+living+cells.">Base editing: precision chemistry on the genome and transcriptome of living cells.</a> Nature Reviews Genetics. 19 (12), 770-788 (2018).</li><li>Spiegel, A., Bachmann, M., Jurado Jim&#233;nez, G., Sarov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-based+knockout+pipeline+for+reverse+genetics+in+mammalian+cell+culture.">CRISPR/Cas9-based knockout pipeline for reverse genetics in mammalian cell culture.</a> Methods. 164-165, 49-58 (2019).</li><li>Suzuki, T., Kazuki, Y., Oshimura, M., Hara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+system+for+simultaneous+or+sequential+integration+of+multiple+gene-loading+vectors+into+a+defined+site+of+a+human+artificial+chromosome.">A novel system for simultaneous or sequential integration of multiple gene-loading vectors into a defined site of a human artificial chromosome.</a> PLoS One. 9, 110404 (2014).</li><li>Bak, R. 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Birincil insan B hücrelerinde hassas genom mühendisliği yakın zamana kadar zorlu olmuştur9,10. Daha önce crispr/cas9 kullanarak birincil insan B hücrelerini tasarlamak için protokoller yayınlamıştık9. Burada, CD19'un verimli KO'suna izin vermek veya EGFP'yivurmak için B hücre yalıtımı, genişletme ve mühendislik için geliştirilmiş protokolleri özetliyoruz.
Burada genişleme protoko...

B hücreleri hematopoetik kök hücrelerden elde edilen lenfosit soyunun bir alt grubudur. Bağışıklık sorunlarına yanıt olarak büyük miktarlarda antikor üreterek adaptif humoral bağışıklık sisteminde kritik bir rol oynarlar1. B hücreleri aynı zamanda bellek B hücrelerinin ve ölümcül olarak farklılaştırılmış, uzun ömürlü plazma hücrelerinin öncüleridir, böylece kalıcı humoral bağışıklıksağlar 2. Plazma hücreleri, özellikle, yıllarca veya onlarca yıl hayatta kalırken belirli bir antikorun büyük miktarlarda üretme yeteneklerinde bağışıklık hücreleri arasında benzersizdir3. Ek olarak, periferik kandan izolasyon kolaylığı, B hücre soyunun yeni hücre bazlı tedaviler için mükemmel bir aday olduğunu yapar4.
Daha önce, lentiviral vektörler veya Uyuyan Güzel transposon kullananlar gibi rastgele entegrasyon yöntemleri, transgene doğum ve ifade 5 , 6,7,8için B hücrelerini tasarlamak için kullanılmıştır. Bununla birlikte, bu yaklaşımların spesifik olmayan doğası, B hücrelerindeki belirli bir genin biyolojik fonksiyonlarının incelenmesini zorlaştırır ve terapötik ortamda eklemeli mutajensis ve değişken transgene ekspresyonu ve / veya susturma riski taşır.
CRISPR/Cas9 sistemi, araştırmacıların çok sayıda türdeki çeşitli hücrelerin genomunu hassas bir şekilde düzenlemelerini sağlayan güçlü bir genom mühendisliği aracıdır. Son zamanlarda, bizimki de dahil olmak üzere iki grup, ex vivo genişleme için yöntemler geliştirdi ve birincil insan B hücrelerinin genom mühendisliğini hedefledi9,10. Birincil insan B hücrelerini lökorezis örneğinden arındırma sürecini açıklayacağız. Bundan sonra, B hücresi genişletme ve izole B hücrelerinin aktivasyonu için güncellenmiş protokolümüzü açıklayacağız. Daha sonra CRISPR/Cas9 mRNA'yı CD19 sgRNA ile birlikte aktif B hücrelerine tanıtmak için elektroporasyon yoluyla belirli bir B hücre reseptörü ve B hücrelerinin ayırt edici bir özelliği olan 19 (CD19) farklılaşma kümesini devirmek için bir süreç açıklayacağız.
CAS9 mRNA çevrilir ve CRISPR/Cas9-sgRNA ribonucleoprotein kompleksi (RNP) oluşturmak için CD19 sgRNA'ya bağlanır. Daha sonra, kompleksteki sgRNA, Cas9'un genin ekson 2'sinde hedef sırada çift iplikçik kırılması (DSB) oluşturmasına neden olur. Hücreler, nükleotidleri tanıtarak veya silerek, frameshift mutasyona yol açarak ve genin bayıltılmasına neden olarak DSB'yi "homolog olmayan uç birleştirme" ile onaracak. Daha sonra akış sitometrisi ile CD19 kaybını ölçecek ve ayrışma (TIDE) analizi ile indellerin takibi ile indel oluşumunu analiz edeceğiz.
Daha sonra CRISPR/Cas9'u, adeno ile ilişkili virüs entegrasyon sitesi 1 (AAVS1) genine gelişmiş yeşil floresan proteinin(EGFP) raya özgü yerleştirilmesine aracılık etmek için rekombinant bir AAV6 vektörü (rAAV6, homolojiye yönelik onarım (HDR) için bir donör şablonu) ile birlikte kullanma sürecini açıklayacağız. AAVS1 geni, bilinen biyolojik işlevleri olmayan aktif bir lokus ve insan genomunda bir AAV viral entegrasyon bölgesidir; bu nedenle, genom mühendisliği için "güvenli bir liman" olarak kabul edilir. Burada, B hücrelerinin genişlemesi ve aktivasyonunun 7 günlük kültürde 44 kata kadar genişlemeye izin verdiğini bildiriyoruz (Şekil 1). B hücrelerinin elektroporasyonunun transeksiyon sonrası 24 saat genel hücre sağlığında(Şekil 2A)hafif bir azalma gösterdiği görüldü. CD19 işaretleyicisinin dağılım grafiği analizi (Şekil 2B) düzenlenen hücrelerde% 83'e kadar azalma gösterdi (Şekil 2C).
Kromatografların GELGIT analizi (Şekil 3A) % indellerin akış sitometrisi ile % protein kaybına benzer olduğunu ortaya koydu (Şekil 3B). KI deneyinin akış sitometrisi analizi, RNP ile birlikte AAV vektörü alan hücrelerin (Şekil 4), % 64'e kadar EGFP pozitif hücreleri (Şekil 5A) ifade ettiğini ve daha sonra kavşak polimeraz zincir reaksiyonu (PCR) ile başarılı entegrasyon gösterdiğini göstermiştir (Şekil 5B). Hücre sayımları, tüm örneklerin mühendislik sonrası 3 gün içinde hızla kurtarıldığunu göstermiştir (Şekil 5C).

<table>
	<tbody>
		<tr>
			<td>Alt-R S.p. Cas9 Nuclease V3 protein, 500 ug</td>
			<td>IDT</td>
			<td>1081059</td>
			<td>smaller size is also available</td>
		</tr>
		<tr>
			<td>Amaxa P3 primary cell 4D- Nucleofector X Kit S (32 RCT)</td>
			<td>Lonza</td>
			<td>V4XP-3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium-chloride-potassium (ACK) lysing buffer</td>
			<td>Quality Biological</td>
			<td>118-156-101</td>
			<td></td>
		</tr>
		<tr>
			<td>CleanCap chemically modified Cas9 mRNA</td>
			<td>Trilink Biotechnology</td>
			<td>L-7206-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>CpG ODN 2006 (ODN 7909) 5 mg</td>
			<td>Invivogen</td>
			<td>TLRL-2006-5</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>Cryostor CS10, 100 mL</td>
			<td>STEMCELL Technology</td>
			<td>7930</td>
			<td></td>
		</tr>
		<tr>
			<td>CTS Immune Cell SR</td>
			<td>Thermo Fisher Scientific</td>
			<td>A2596101</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep human B cells isolation kit</td>
			<td>STEMCELL Technology</td>
			<td>17954</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience fixable viability dye eFlour 780</td>
			<td>eBiosciences</td>
			<td>65-0865-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Excellerate B cell media, Xeno-free</td>
			<td>R&#38;D Systems</td>
			<td>CCM031</td>
			<td>B-cell basal medium</td>
		</tr>
		<tr>
			<td>Falcon 14 mL Polypropylene Round-bottom Tube</td>
			<td>Corning</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>R&#38;D Systems</td>
			<td>S11550</td>
			<td>For thawing B cells only</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneMate SnapStrip&#174; 8-Strip 0.2 mL PCR Tubes with Individual Attached Dome Caps</td>
			<td>BioExpress</td>
			<td>T-3035-1 / 490003-692</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone 0.0067M PBS (No Ca, No Mg) or 1x PBS</td>
			<td>GE lifesciences</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza 4D Nucleofector core unit</td>
			<td>Lonza</td>
			<td>AAF-1002B</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza 4D Nucleofector X unit</td>
			<td>Lonza</td>
			<td>AAF-1002X</td>
			<td></td>
		</tr>
		<tr>
			<td>Mega CD40 Ligand</td>
			<td>Enzo Life Sciences</td>
			<td>ALX-522-110-C010</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty</td>
			<td>Sigma-Aldrich</td>
			<td>C1562-1EA</td>
			<td>For freezing cells</td>
		</tr>
		<tr>
			<td>Pen/Strep 100X</td>
			<td>Sigma-Aldrich</td>
			<td>TMS-AB2-C</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP anti-human CD19 clone HIB19</td>
			<td>biolegend</td>
			<td>302228</td>
			<td>smaller size is also available</td>
		</tr>
		<tr>
			<td>rAAV6 SA-GFP pakaging ( with our SA-GFP cassette see Figure 4.)</td>
			<td>Vigene Biosciences</td>
			<td>N/A</td>
			<td>large scale packaging, 1e13 GC/mL, 500 mL</td>
		</tr>
		<tr>
			<td>Recombinant human IL-10 protein 250 ug</td>
			<td>R&#38;D Systems</td>
			<td>217-IL-250</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>Recombinant human IL-15 protein 25 ug</td>
			<td>R&#38;D Systems</td>
			<td>247-ILB-025</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>The Big Easy EasySep Magnet</td>
			<td>STEMCELL Technology</td>
			<td>18001</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>Tris-EDTA (TE) buffer</td>
			<td>Fisher Scientific</td>
			<td>BP2476100</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome engineering of primary human b cells using crispr/cas9

B hücreleri hematopoetik kök hücrelerden elde edilen lenfositlerdir ve adaptif bağışıklık sisteminin humoral kolunun önemli bir bileşenidir. Periferik kandan izolasyon kolaylığı, in vitroyu genişletme yetenekleri ve in vivo olarak uzun ömürlü olmaları nedeniyle hücre bazlı tedaviler için çekici adaylar yaparlar. Ek olarak, normal biyolojik işlevleri -büyük miktarlarda antikor üretmek için- enfeksiyonla savaşmak için rekombinant antikor veya enzimopatiklerin tedavisi için bir enzim gibi çok büyük miktarlarda terapötik proteini ifade etmek için kullanılabilir. Burada, primer insan B hücrelerini periferik kan mononükleer hücrelerinden (PBMC) izole etmek ve izole edilmiş B hücrelerini in vitro olarak etkinleştirmek / genişletmek için ayrıntılı yöntemler sunuyoruz. Daha sonra B hücrelerindeki endojen genlerin bölgeye özgü KO'su için CRISPR/Cas9 sisteminin kullanılmasıyla ilgili adımları gösteriyoruz. Bu yöntem, ilgi çekici genlerin biyolojik işlevlerini incelemek için kullanılabilecek çeşitli genlerin verimli KO'suna izin verir. Daha sonra CRISPR/Cas9 sistemini kullanma adımlarını, B hücrelerindeki transgene ifade kasetinin raya özgü verimli entegrasyonu için rekombinant, adeno ilişkili, viral (rAAV) vektör ile birlikte gösteriyoruz. Birlikte, bu protokol, birincil insan B hücrelerinde genlerin biyolojik işlevlerini incelemek ve B hücre terapötiklerinin geliştirilmesi için kullanılabilecek adım adım bir mühendislik platformu sağlar.

Güncellenmiş genişletme ve aktivasyon protokolü, B hücrelerinin 7 günde 44 kata kadar hızlı genişlemesini sağladı(Şekil 1; n =3 donörler). KO deneyinde, Trypan mavi boyama kullanan B hücre sayısı, hem kontrolde hem de CD19 KO örneklerinde 24 saat post-elektroporasyonda hücre iyileşmesinde hafif bir azalma ile% 80'den fazla uygulanabilir hücre gösterdi(Şekil 2A; p ≥ 0.05, n = 3 donör). Bu sonuç, elektroporasyonun genel B hücre sağlığ...

Watch this Scientific Journal Video about Genome Engineering of Primary Human B Cells Using CRISPR/Cas9 at JoVE.com

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

Burada, B hücrelerindeki genlerin biyolojik fonksiyonlarını ve B hücre terapötiklerinin gelişimini incelemek için gen nakavt (KO) ve knock-in (KI) için birincil insan B hücrelerinin CRISPR/ Cas9 tabanlı genom mühendisliği için ayrıntılı, adım adım bir protokol sunuyoruz.

CRISPR/Cas9 Kullanarak Birincil İnsan B Hücrelerinin Genom Mühendisliği

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

This protocol allows researchers to precisely eliminate a gene in B cells to study the loss of function effects of that gene or to insert a trans gene for stable trans gene expression that can be used for research or therapeutic purposes. The main advantage of this method is that it provides a universal platform that makes it possible to easily and precisely engineer B cells with high efficiencies. This method can be used to engineer B cells to express recombinant antibodies or enzymes and then transfer those B cells to treat infections or in enzymopathies.To begin, thaw B cells in a 37 degree Celsius water bath. While waiting, transfer two milliliters of pre warmed FBS into a sterile, 15-milliliter conical tube. Once the B cells are completely thawed, immediately add one milliliter of pre-warmed FBS, drop-wise, into the sample.Incubate at room temperature for one minute. Gently pipette to resuspend the sample and transfer the whole volume, drop-wise, into a conical tube containing two milliliters of prewarmed FBS. Bring the volume to 15 milliliters with sterile PBS, then cap the tube and invert it gently two to three times.Centrifuge the sample at 400 times G for five minutes, then discard the supernatant without disturbing the cell pellet. Resuspend the pellet with one milliliter of pre-equilibrated B-cell expansion medium and count the cells. The total cell number should be approximately 10 to the seventh cells.Transfer the cells into a flask containing 20 milliliters of the pre-equilibrated B-cell expansion medium. The final concentration of the cells, should be approximately five times 10 to the fifth cells per milliliter. Incubate the flask vertically in a tissue culture incubator.Prepare the CRISPR/Cas9 transfecting substrate by mixing one microgram of chemically modified sgRNA with 1.5 micrograms of chemically modified streptococcus pyogenes Cas9 nucleus mRNA per transfection reaction. For control, add one microliter of TE buffer, instead of sgRNA, into a 0.2-milliliter tube of an eight tube strip. Turn on an electroporator and prepare the nuclear infection reagents.Count and transfer 1 million B cells per transfection reaction, into a sterile conical tube. Bring the volume to 15 milliliters with sterile PBS and centrifuge at 400 times G for five minutes. After centrifugation, discard the supernatant.Resuspend the cells with 10 milliliters of sterile PBS and centrifuge at 400 times G for five minutes, then discard the supernatant completely without disturbing the cell pellet. Add 0.5 micrograms of chemically modified GFP mRNA per 1 million B cells to the cell pellet. Resuspend the pellet with 20 microliters of primary cell transfection reagent per 1 million B cells and mix gently by pipetting five to six times.Transfer 20.5 microliters per transfection reaction into the 0.2-milliliter tubes containing CRISPR/Cas9 reagent. Pipette up and down once to mix and transfer the entire volume into a transfection cuvette. Cap and tap the cuvette on the bench gently to ensure that the liquid covers the bottom of the cuvette.Use the human primary B-cell protocol on the electroporator for transfection. Rest the electroporated cells in the cuvette at room temperature for 15 minutes. Then transfer 80 microliters of the pre-equilibrated B-cell expansion medium into the transfection reaction in the cuvette.Place the cuvette in the tissue culture incubator for 30 minutes. Gently pipe head a couple of times to mix and transfer the whole volume of the sample from the cuvette to an appropriate well of a 48-well tissue culture plate containing one milliliter of the B-cell expansion medium. The final concentration of the cells should be 1 million cells per milliliter.Performing a gene knock-in experiment, transfer recombinant adeno associated type-six viral vector at 500, 000 multiplicity of infection into the appropriate well containing electroporated cells. Place the plate in a tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide with humidity. In the knockout experiment, the B-cell count showed more than 80%viable cells with a slight reduction in cell recovery in both the control and the CD19 knockout samples at 24 hours post electroporation.B cells were collected on day five post-transfection for flow cytometry and tide analysis. Representative scatterplots of the control and knockout samples showed 14 and 95%CD19 negative cells respectively, demonstrating a significant reduction in CD19 expression in the knockout samples. Chromatograms of genomic sequencing showed double peaks in the CD19 knockout B cells, indicating insertions or deletions of nucleotides post-CRISPR/Cas9 mediated double-stranded breaks.Indel analysis of the chromatographs of the knockout samples showed a high percent of indel formation at the CD19 locus, which is consistent with percent CD19 protein loss detected by flow cytometry. B cells from the knock-in experiment were collected on day 12, post engineering. Scatterplots showed 64%of EGFP positive cells in the sample that received the recombinant adeno associated type-six viral vector, together with RNP, whereas none or minimal EGFP positivity was observed in the control and vector-only samples respectively.A junction PCR amplification, showed 1.5 kilobase pair amplicons in the knock-in sample and no PCR product was observed in either the control or vector-only sample. Cell counts showed that the engineering process affects cell recovery in the knock-in sample more than the control or the vector-only samples. However, all samples quickly rebounded within three days after engineering.This procedure can also be used to introduce base editor agents to alter single base pairs in order to induce or correct single point mutations in the B-cell genome. It could also be used to introduce premature stop codons or to alter splice sites, allowing for multiple gene knockouts without the risk of chromosomal translocations. Before this technique was developed, engineering human B cells would inquire the use of other more challenging gene engineering approaches.This technique is an easier, less expensive and quicker alternative. It could also potentially be used to synthesize cell therapy products for clinical use.

genome-engineering-of-primary-human-b-cells-using-crisprcas9

Research

JoVE Journal

Biology

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Yetişkin Zebra balığı İç Kulak Duyu Epiteli Brüt ve Güzel Diseksiyon ( Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Biyoloji

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

İnsan CD40 aktive B hücreleri üretilmesi

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

İlköğretim İnsan fibroblast Kültürler gelen naif Erişkin Kök Hücre İzolasyon

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Tasarımcı nükleazlar kullanılması Fare Genom Mühendisliği

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

CRISPR / Cas9 üzerinden Memeli Hücre Hatlarında Genomik Delesyonlar Üretimi

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

Tüm cep telefonu Elektrofizyoloji birincil kültürlü fare Enterochromaffin hücre

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

Kıvılcım Susturma: CRISPR/Cas9 Zayıf Elektrikli Balıklarda Genom Düzenleme

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

CRISPR/Cas9 Kullanarak Boş Mutasyonlar Oluşturmak için Culex Sivrisineklerinin Embriyolarının Hazırlanması ve EnjekteSi

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

CRISPR/Cas9 Teknolojisi Kullanılarak Göçmen Locust'un Homozigot Mutantlarının İnşası

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.