Name: stem cell-derived viral ag-specific t lymphocytes suppress hbv replication in mice
Uploaded: 2019-09-25T13:30:00
Description: Watch this Scientific Journal Video about Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice at JoVE.com

Yazarlar HBs183-91 (s183) (FLLTRILTI) için cDNA sağlamak için Toronto Genel Hastanesi Araştırma Enstitüsü'nden Dr Adam J Gehring teşekkür- özel A2-sınırlı insan-murine hibrid TCR genleri, ve Dr Pei-Jer Chen Ulusal Tayvan Üniversitesi sağlamak için pAAV/HBV 1.2 yapısı. Bu çalışma Ulusal Sağlık Enstitüsü Hibe R01AI121180, R01CA221867 ve R21AI109239 J. S tarafından desteklenmiştir.

Tüm hayvan deneyleri Texas A&M Üniversitesi Hayvan Bakım Komitesi (IACUC; #2018-0006) tarafından onaylanır ve Laboratuvar Hayvan Bakımı Nın Değerlendirilmesi ve Akreditasyonu Derneği'nin yönergelerine uygun olarak yürütülmektedir. Fareler 6-9 yaş ları arasında kullanılır.
1. IPSC'lerden viral Ag'ye özgü CD8+ T hücrelerinin (iPSC-CD8+ T hücreleri) üretimi
<ol><li>Retroviral yapılar oluşturulması NOT: TCR α ve β genleri 2A kendi kendine ayr...

<ol>
	<li>Scaglione, S. J., Lok, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+hepatitis+B+treatment+in+clinical+practice.">Effectiveness of hepatitis B treatment in clinical practice.</a> Gastroenterology. 142 (6), 1360-1368 (2012).</li><li>Osiowy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+infancy+and+beyond...+ensuring+a+lifetime+of+hepatitis+B+virus+(HBV)+vaccine-induced+immunity.">From infancy and beyond... ensuring a lifetime of hepatitis B virus (HBV) vaccine-induced immunity.</a> Human Vaccines &amp; Immunotherapeutics. 14 (8), 2093-2097 (2018).</li><li>Gish, R. G., 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=Loss+of+HBsAg+antigen+during+treatment+with+entecavir+or+lamivudine+in+nucleoside-naive+HBeAg-positive+patients+with+chronic+hepatitis+B.">Loss of HBsAg antigen during treatment with entecavir or lamivudine in nucleoside-naive HBeAg-positive patients with chronic hepatitis B.</a> Journal of Viral Hepatitis. 17 (1), 16-22 (2010).</li><li>Kurktschiev, P. D., 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=Dysfunctional+CD8++T+cells+in+hepatitis+B+and+C+are+characterized+by+a+lack+of+antigen-specific+T-bet+induction.">Dysfunctional CD8+ T cells in hepatitis B and C are characterized by a lack of antigen-specific T-bet induction.</a> Journal of Experimental Medicine. 211 (10), 2047-2059 (2014).</li><li>Fisicaro, P., 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=Antiviral+intrahepatic+T-cell+responses+can+be+restored+by+blocking+programmed+death-1+pathway+in+chronic+hepatitis+B.">Antiviral intrahepatic T-cell responses can be restored by blocking programmed death-1 pathway in chronic hepatitis B.</a> Gastroenterology. 138 (2), 682-693 (2010).</li><li>Schurich, A., 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=The+third+signal+cytokine+IL-12+rescues+the+anti-viral+function+of+exhausted+HBV-specific+CD8+T+cells.">The third signal cytokine IL-12 rescues the anti-viral function of exhausted HBV-specific CD8 T cells.</a> PLoS Pathogens. 9 (3), 1003208 (2013).</li><li>Gehring, A. J., 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+virus-specific+T+cells+that+target+HBV+infected+hepatocytes+and+hepatocellular+carcinoma+cell+lines.">Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines.</a> Journal of Hepatology. 55 (1), 103-110 (2011).</li><li>Xia, Y., 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=Interferon-gamma+and+Tumor+Necrosis+Factor-alpha+Produced+by+T+Cells+Reduce+the+HBV+Persistence+Form,+cccDNA,+Without+Cytolysis.">Interferon-gamma and Tumor Necrosis Factor-alpha Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis.</a> Gastroenterology. 150 (1), 194-205 (2016).</li><li>Huang, L. R., Wu, H. L., Chen, P. J., Chen, D. 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S., 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=Human+effector+CD8++T+cells+derived+from+naive+rather+than+memory+subsets+possess+superior+traits+for+adoptive+immunotherapy.">Human effector CD8+ T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy.</a> Blood. 117 (3), 808-814 (2011).</li><li>Kerkar, S. 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D., Themeli, M., Sadelain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-1-+and+CTLA-4-based+inhibitory+chimeric+antigen+receptors+(iCARs)+divert+off-target+immunotherapy+responses.">PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses.</a> Science Translational Medicine. 5 (215), (2013).</li><li>Maus, M. 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Bu protokol, bir murine modelinde HBV replikasyonlarını bastırmak için ACT olarak kullanılmak üzere viral Ag-spesifik iPSC-CTLs oluşturmak için bir yöntem sunar. Kronik HBV enfeksiyonunda viral genom kararlı bir mini kromozom oluşturur, hepatositin ömrü boyunca devam edebilen kovalent kapalı dairesel DNA (cccDNA). Viral mini kromozomun temizlenmesinin hedeflanması kronik HBV enfeksiyonunun tedavisine neden olabilir. Mevcut antiviral tedavi virüsün ters transkriptazını hedefler ancak nadiren cccDNA tara...

Akut enfeksiyon dan sonra, adaptif bağışıklık sistemi (yani, humoral ve hücresel bağışıklık) akut HBV ile ilgili hepatit toplu kontrol eder. Yine de, HBV-endemik bölgelerde insanların bir dizi virüsleri ortadan kaldırmak ve daha sonra kronik bireyler olarak dönüştürmek olamaz. Kronik hastaların %25'inden fazlası (>250 milyon kişi) dünya çapında ilerleyici karaciğer hastalığı gelişir ve karaciğer sirozu ve/veya hepatosellüler karsinom (HCC)1. Sonuç olarak, ısrarla enfekte hücrelerin eradikasyonu genel bir sağlık sorunu olmaya devam etmektedir, mevcut bir aşı olmasına rağmen2 ve çok sayıda antiviral ilaçlar geliştirilmektedir. HBV enfeksiyonu için standart tedavi IFN-α, nükleozit ve nükleotid analogları içerir. Bu ajanlar doğrudan antiviral aktivite ve bağışıklık modülatör kapasiteleri var. Bununla birlikte, HBe antijeninin (Ag)+ anti-HBe antikor (Ab) ve serum HBV deoksiribonükleik asit (DNA) kaybı ile birlikte seropdönüşümü tedavi edilen hastaların yaklaşık %20'sinde ayrı ayrı görünür ve virüsün tüm immünolojik kontrolü HBsAg yoksunluğu ile doğrulanan en fazla% 53. Ayrıca, tedaviye yanıt genellikle dayanıklı değildir. Rekombinant HBs Ag ile profilaktik aşılama enfeksiyonu önlemede son derece etkilidir, ancak terapötik HBs Ag aşısı etkili değildir. Açıkçası, T hücre aracılı bağışıklık yanıtları HBV enfeksiyonu ve karaciğer bozukluğu kontrolünde kritik bir rol oynamaktadır; ancak, kronik hepatit hastalarında, HBV-reaktif T hücreleri genellikle silinir, işlevsiz, ya da tükenmiş dönüştürmek4,5,6. Sonuç olarak, kalıcı HBV enfeksiyonu olan bireylerde, anti-viral ilaç, immüno-modüler sitokinler veya küratif bağışıklama yoluyla HBV'ye özgü bağışıklığı (yani T hücre tabanlı bağışıklık) geri getirme girişimleri başarıya ulaşamaz.
HBV Ag-spesifik T hücrelerinin benimseyen hücre transferi (ACT) sonunda kalan hepatositler wih HBV7ortadan kaldırmak için yönlendirilen etkili bir tedavidir,8. HBV'ye özgü KTK'ların HBV enfekte olmuş farelere aktiretinin geçici, hafif hepatite ve hepatositlerde HBV ribonükleik asit (RNA) transkriptlerinde dramatik bir düşüşe neden olduğu gösterilmiştir. Bu çalışmalarda, CTLs HBV genlerin transkripsiyonunu inhibe etmedi ama HBV transkript bozulması gelişmiş9. HBV'ye özgü CtL'ler viral enfeksiyonu önlemek ve HBV10,11'intemizlenmesine aracılık etmek için önemlidir. ACT tabanlı ilaçlar için, in vivo resettlement için yüksek reaktivite ile HBV özgü T hücrelerinin in vitro genişleme ideal bir yöntem olduğu ileri sürülmüştür12,13,14; Bununla birlikte, mevcut yaklaşımlar potansiyel tedaviler için hastalardan HBV'ye özgü T hücrelerinin uygun miktar ve niteliklerini üretme, ayırma ve büyütme yetenekleri açısından kısıtlanmıştır.
Klinik çalışmalarda HBV virüs bulaşmış hepatositlere özgü mühendislik T hücreleri vasıtası ile hücre bazlı tedavilerin güvenliği, pratikliği ve prospektif terapötik aktivitesi mevcut olsa da, olumsuz etkileri hakkında endişeler vardır yanlış eşleşmeli T hücre reseptöründen çapraz reaktivite nedeniyle otoimmün yanıtlardan kaynaklanan (TCR)15,16, non-spesifik TCR17 tarafından hedef dışı Ag tanıma ve bir şierik Ag reseptörü (CAR) tarafından hedef dışı toksisite 18.000 , Sağlıklı dokularla 19. Şu anda, genetiği değiştirilmiş T hücreleri, sadece in vivo kısa süreli kalıcılık var, genellikle ara veya daha sonra etkili T hücreleridir. Bugüne kadar, pluripotent kök hücreler (PSCs) naif tek tip Ag-spesifik T hücreleri20, 21,22,23yüksek sayıda üretmek için kullanılabilir tek kaynaktır. İndüklenen PSC'ler (iPSC'ler) sadece birkaç transkripsiyon faktörünün gen transdüksiyonu yoluyla hastanın somatik hücrelerinden dönüştürülür. Sonuç olarak, iPSC'ler embriyonik kök hücreler (ESCs)24benzer özelliklere sahip. Doku replasmanına ek olarak, doku replasmanına ek olarak, kendini yenileme esnekliği ve olasılığı sayesinde, iPSC tabanlı tedaviler rejeneratif tıpta yaygın olarak uygulanabilir. Ayrıca, iPSCs altında yatan alaylar önemli ölçüde mevcut hücre tabanlı tedavileri artırabilir.
Bu yöntemin genel amacı, ACT tabanlı immünoterapi için iPSC'lerden (yani iPSC-CTLs) hbv'ye özgü ctl'ler üretmektir. Alternatif tekniklere göre avantajları HBV'ye özgü iPSC-CTL'lerin tek tip TCR ve naif fenotip olmasıdır, bu da ACT'den sonra daha fazla bellek T hücre gelişimiile sonuçlanır. HBV'ye özgü iPSC-CTL'lerin AKTIV'sinin karaciğerde fonksiyonel CD8+ T hücrelerinin geçişini artırdığı ve hem karaciğerlerde hem de yönetilen farelerin kanlarında HBV replikasyonlarını azalttığı gösterilmiştir. Bu yöntem HBV immünoterapi için viral Ag-spesifik iPSC-CTLs potansiyel kullanımını ortaya koymaktadır ve viral immünoterapi için diğer viral Ag-spesifik iPSC-T hücreleri oluşturmak için adapte edilebilir.

<table>
	<tbody>
		<tr>
			<td>HHD mice</td>
			<td>Institut Pasteur, Paris, France</td>
			<td></td>
			<td>H-2 class I knockout, HLA-A2.1-transgenic (HHD) mice</td>
		</tr>
		<tr>
			<td>iPS-MEF-Ng-20D-17</td>
			<td>RIKEN Cell Bank</td>
			<td>APS0001</td>
			<td></td>
		</tr>
		<tr>
			<td>SNL76/7</td>
			<td>ATCC</td>
			<td>SCRC-1049</td>
			<td></td>
		</tr>
		<tr>
			<td>OP9</td>
			<td>ATCC</td>
			<td>CRL-2749</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV/HBV1.2 plasmid</td>
			<td>Dr. Dr. Pei-Jer Chen (National Taiwan University Hospital, Taiwan)</td>
			<td></td>
			<td>HBV DNA construct</td>
		</tr>
		<tr>
			<td>HBs183-91(s183) (FLLTRILTI)-specific TCR genes</td>
			<td>Dr. Adam J Gehring (Toronto General Hospital Research Institute, Toronto, Canada)</td>
			<td></td>
			<td>FLLTRILTI-specific A2-restricted human-murine hybrid TCR genes (V&#945;34 and V&#946;28)</td>
		</tr>
		<tr>
			<td>OVA257&#8211;264-specific TCR genes</td>
			<td>Dr. Dario A. Vignali (University of Pittsburgh, PA)</td>
			<td></td>
			<td>SIINFEKL-specific H-2Kb-restricted TCR genes</td>
		</tr>
		<tr>
			<td>Anti-CD3 (17A2) antibody</td>
			<td>Biolegend</td>
			<td>100236</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD44 (IM7) antibody</td>
			<td>BD Pharmingen</td>
			<td>103012</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD4 (GK1.5) antibody</td>
			<td>Biolegend</td>
			<td>100408</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD8 (53-6.7) antibody</td>
			<td>Biolegend</td>
			<td>100732</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-IFN-&#947; (XMG1.2) antibody</td>
			<td>Biolegend</td>
			<td>505810</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TNF-a (MP6-XT22) antibody</td>
			<td>Biolegend</td>
			<td>506306</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Invitrogen</td>
			<td>A10490-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-HBs antibody</td>
			<td>Thermo Fisher</td>
			<td>MA5-13059</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysis buffer</td>
			<td>Lonza</td>
			<td>10-548E</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>Sigma</td>
			<td>B7651</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>ABCD1234</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH3007.01</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria Fusion cell sorter</td>
			<td>BD</td>
			<td>656700</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>MilliporeSigma</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneJammer</td>
			<td>Agilent</td>
			<td>204130</td>
			<td></td>
		</tr>
		<tr>
			<td>HLA-A201-HBs183-91-PE pentamer</td>
			<td>Proimmune</td>
			<td>F027-4A - 27</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP Anti-Mouse Secondary Antibody</td>
			<td>Invitrogen</td>
			<td>A27025</td>
			<td></td>
		</tr>
		<tr>
			<td>mFlt-3L</td>
			<td>Peprotech</td>
			<td>250-31L</td>
			<td></td>
		</tr>
		<tr>
			<td>mIL-7</td>
			<td>Peprotech</td>
			<td>217-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease S7</td>
			<td>Roche</td>
			<td>10107921001</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>MilliporeSigma</td>
			<td>P6148-500G</td>
			<td>Caution: Allergenic, Carcenogenic, Toxic</td>
		</tr>
		<tr>
			<td>Permeabilization buffer</td>
			<td>Biolegend</td>
			<td>421002</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>MilliporeSigma</td>
			<td>107689</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong&#8482; Gold Antifade Mountant with DAPI</td>
			<td>Invitrogen</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp MinElute Virus Spin Kit</td>
			<td>Qiagen</td>
			<td>57704</td>
			<td></td>
		</tr>
	</tbody>
</table>

stem cell-derived viral ag-specific t lymphocytes suppress hbv replication in mice

Hepatit B virüsü (HBV) enfeksiyonu küresel bir sağlık sorunudur. Dünya çapında etkilenen 350 milyondan fazla kişi ile, HBV enfeksiyonu karaciğer kanserinin önde gelen nedeni olmaya devam etmektedir. Bu, özellikle gelişmekte olan ülkelerde önemli bir sorundur. Bağışıklık sisteminin HBV'ye karşı etkili bir yanıt vermemesi kronik enfeksiyona yol açar. HBV aşısı mevcut ve yeni antiviral ilaçlar oluşturulmaktadır rağmen, virüs-rezervuar hücrelerinin eradikasyonu önemli bir sağlık konusu olmaya devam etmektedir. Burada açıklanan viral antijen üretimi için bir yöntemdir (Ag) -spesifik CD8+ sitotoksik T lenfositler (CTLs) indüklenen pluripotent kök hücrelerden türetilen (iPSCs) (yani, iPSC-CTLs), HBV replikasyonbastırmak için yeteneğine sahip. HBV replikasyonu farelerde hbv ekspresyonu plazmidi pAAV/HBV1.2'nin karaciğere hidrodinamik enjeksiyonu ile etkin bir şekilde indüklenir. Daha sonra, HBV yüzey Ag-spesifik fare iPSC-CTLs büyük ölçüde karaciğer ve kanda HBV replikasyon bastırır ve hepatositlerde HBV yüzey Ag ekspresyonunu önler, benimsenden aktarılır. Bu yöntem, hidrodinamik enjeksiyon sonrası farelerde HBV replikasyonu gösterir ve kök hücre kaynaklı viral Ag-spesifik CTLs HBV replikasyonbastırmak olabilir. Bu protokol HBV immünoterapi için yararlı bir yöntem sağlar.

Burada gösterildiği gibi, HBV viral Ag-spesifik iPSC-CD8+ T hücreleri in vitro kültür sistemi tarafından oluşturulur. Bu viral Ag-spesifik iPSC-CD8+ T hücrelerinin ACT sonra önemli ölçüde bir murine modelinde HBV replikasyon bastırmak(Ek Dosya 1). Fare iPSC bir insan-fare hibrid HBV TCR geni kodlama MIDR retroviral yapı ile transdükmen edilir (HBs183-191-spesifik, s183), sonra gen-transdükse iPSC'ler notch ligands (hem DL1 ve DL4) ifade OP9-DL1/DL4 hücrel...

Watch this Scientific Journal Video about Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice at JoVE.com

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Burada sunulan kök hücre kaynaklı viral antijen (Ag) özgü T lenfositlerin benimseyen hücre transferi (ACT) kullanarak farelerde hepatit B virüsü (HBV) replikasyonu nun etkin bastırılması için bir protokoldür. Bu işlem HBV enfeksiyonunun act tabanlı potansiyel immünoterapisi için uyarlanabilir.

Kök Hücre Kaynaklı Viral Ag-Spesifik T Lenfositler Farelerde HBV Replikasyonlarını Baskılar

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...

Bu yöntem farelerde HBV replikasyonunun etkin bastırılması için iPSC kaynaklı viral antijen spesifik T hücresinin benimseyen hücre transferinde anahtar soruların yanıtlanmasına yardımcı olabilir. Bu tekniğin en büyük avantajı, iPSC kaynaklı viral antijene özgü T hücrelerinin tek tip T hücre reseptörüne ve naif fenotiplere sahip olmasıdır. HBV'ye özgü iPSC CD8-pozitif T hücrelerinin farklılaşması için, plaka s183 T hücre reseptör gen-transdükre edilmiş iPSC'ler op9-DL1/DL4 monolayer alfa-minimum ortamda 20% fetal sığır serum ve murine Flt3 ligand ile desteklenen bir hücre kültürü inkübatör kendi co-kültür için.Hücre morfolojisini değerlendirmek için ortak kültürü düzenli olarak izleyin. 28. günde, süpernatant ve yüzen hücreleri çıkarın ve %0,25 tripsin ile yapışık hücreleri ayırın. Hücreler kültür kabı nın altından kalktığında, sekiz mililitre iPSC orta sıyla enzimatik reaksiyonu durdurun ve hücreleri santrifüj le toplayın.Taze orta 10 mililitre pelet resuspend. Hücreleri bir iPSC kaynaklı CD8-pozitif T hücresine seyreltin ve hücre kültürü kuluçka makinesinde 40 saatlik bir kuluçka için dört s183 peptid-darbeli splenosit oranına ayarlayın. Son yedi saat içinde, hücre aktivasyonu sırasında taşıma süreçlerini engellemek için kültüre seyreltilmiş brefeldin A dört mikrolitre ekleyin.Standart protokollere göre ilgi hücre içi sitokinlerin akış sitometrik analizi için hücreleri lekeleyin. Kuyruk damarı ile hidrodinamik HBV plazmid iletimi için, PBS vücut kütlesinin% 8 eşdeğer HBV plazmid 10 mikrogram seyreltin. Plazmidi fare başına 26-gauge, 1/2-inç iğne ile donatılmış bir üç mililitrelik şırınga yükleyin.Kuyruk damarlarını genişletmek için BEŞ dakika boyunca bir ısı lambası altında Isı HHD fareler, ve dikkatle plastik bir restrainer içine bir fare çekin. Kuyruğun orta üçte bir dilate lateral kuyruk damarı bulmak ve üç-beş saniyelik bir süre boyunca kuyruk ven ile plazmid tüm hacmi teslim etmek için ven paralel iğne ekleyin. Viral antijene özgü iPSC kaynaklı CD8-pozitif T hücre benimseyen hücre transferi için, gösterildiği gibi kültürün 22.Hücre kültürü kuluçka makinesinde 30 dakika kaldıktan sonra yüzen hücreleri 70 mikrometrelik bir naylon süzgeçten filtreleyin ve canlı hücreleri sayın. PBS konsantrasyonu mililitre başına yedi hücre için 1,5 kez 10 hücreleri seyreltin. Dört ila altı haftalık HHD farelerine 200 mikrolitre hücre enjekte edin, az önce gösterildiği gibi kuyruk damarına.Hücre transferinden sonra 14. Enfeksiyon sonrası uygun deneysel uç noktalarda, her enfekte hayvandan karaciğeri hasat edin ve karaciğerleri 0,5 by-0,5 by-0,3 santimetrelik doku örnekleri ne kadar kesin. Örnekleri 4-24 saat boyunca %10 nötr tampon formaline yerleştirin, ardından 2,5 molar formik asitte altı ila 24 saat kireçlenme yi takip edin.Daha sonra, örnekleri üç dakika boyunca ksilen içinde durulayın, ardından %100 etanol, %95 etanol ve deiyonize suyla iki dakikalık durulama. Son su yıkamadan sonra, 90 santigrat derece bir milimolar EDTA dokuları dekalsite, oda sıcaklığında 30 dakikalık kuluçka izledi. Doku soğuduğunda, pbs'deki örnekleri dört dakika durulayın ve numuneleri ayırmak ve yeniden sulandırmak için ksilen ve etanol kullanın.İmmünofloresan etiketleme için, 75 ila %100 nemlendirilmiş bir odada oda sıcaklığında iki saat boyunca 200 mikrolitre HBV yüzey antijene özgü antikor ile kesitleri lekelayın. Kuluçka süresinin sonunda, numuneleri her yıkama için taze PBS'de beş adet beş dakikalık yıkayarak yıkayın. Floresan mikroskopi ile görüntüleme için slaytları kapaklı dudaklarla takmadan önce hücreleri standart protokollere göre DAPI ile etiketleyin.İmmünohistokimyasal analiz için, standart protokollere göre hematoksilin ve eozin ile kesitleri lekeleyin ve ışık mikroskobu ile inflamatuar hücrelerin karaciğere infiltrasyonunu değerlendirin. iPSC türetilmiş CD3-pozitif CD8-pozitif popülasyonun akış sitometrik analizi, HBV T hücre reseptör transdüksiyonunun viral s183-spesifik CD8-pozitif T hücrelerinin oluşumunu önemli ölçüde artırdığını ortaya koymaktadır. S183 peptid ile darbeli T-tükenmiş splenositler ile uyarılmasından sonra, CD4-negatif CD8 tek pozitif iPSC türetilmiş T hücreleri hücre içi akış sitometrik analizi ve ELISA tarafından tespit edilen büyük miktarda interferon gama üretirler.Hidrodinamik enjeksiyon ile HBV enfeksiyonu sonra, DNA replikasyonu gün altı zirveleri ve yavaş yavaş azalır, gerçek zamanlı PCR analizi ile gözlenen. Ayrıca, viral replikasyon önemli ölçüde kontrol hücreleri alan farelere göre HBV viral antijenik spesifik T hücreleri alan farelerde her zaman noktalarında azalır. HBV yüzey proteini de önemli ölçüde kontrol hücreleri ile tedavi farelere göre HBV viral antijen-spesifik pre-iPSC sitotoksik lenfositler alan farelerde azalır, açıkça viral antijene özgü iPSC CD8-pozitif T hücrelerinin bir murine modelinde HBV replikasyonazaltmak için yeteneğine sahip olduğunu gösteren.Şimdi nasıl benimseyen immünoterapi için iPSC viral antijen-spesifik T hücreleri oluşturmak için iyi bir anlayışa sahip olmalıdır.

stem-cell-derived-viral-ag-specific-t-lymphocytes-suppress-hbv

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Fare T-hücrelerinin, T-hücre reseptörleri retroviral İletimi

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

İmmünoloji ve Enfeksiyon

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Sitotoksik T Lenfositler Beta Hücre Ölümü aracılığı ile değerlendirin yöntemleri

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus-spesifik T hücrelerinin üretilmesi Allojenik Hematopoetik Kök Hücre Nakli sonra Viral Enfeksiyonlar önleme / tedavi etmek için

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Kordon Kanı adlı Sitotoksik T lenfositler genişletilmesi olduğunu Hedef Sitomegalovirüs, Epstein-Barr Virus ve Adenovirüs

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

T Lenfositler doğru İsteyerek Pluripotent Kök Hücrelerinin Yönetmen Farklılaşma

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

İzolasyon ve naif CD4 T Lenfositler Th17 Farklılaşma

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T Lenfositlerindeki Integrin Aktivasyonunun Çalışması için statik Adezyon Deneyi

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

Adenovirüs ile enfekte Normal İnsan Hücreleri Viral Çoğaltma Bölme zenginleştirilmiş Alt nükleer Kesirler izolasyonu

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

İnsan Palatine Bademcikler B ve T Lenfosit için Verimli İzolasyon Protokolü

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Raportör fareler kullanılarak IL-22 eksprese eden lenfositlerin Görselleştirme

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...