<ol><li> 3-5x10 6 hücre / 6 cm doku cultureplate dönüşümü, plaka 293T hücreleri önce akşam. Not: Biz transfeksiyon ve efficacyas virüs üreten hücrelerin kendi kolaylığı için 293T hücreleri kullanır. Bu hücreler, plazmid bir yardımcı tanıtıldı sürece virüs ambalaj eksikliği vardır . </li><li> Sabah ilk, orta hafifçe kaldırın ve 4 ml 293T hücre med 25 mM Klorokin içeren değiştirin. 1 saat için kuvöz koyun. Not: plaka konfluent yaklaşık% 80 olmalıdır. </li><li> Bu arada, 5 ml tüpler, bir seferde 2 plakaları için virüs yapımı karışımının hazırlanması: <ol><li> 2 x 10 mg retroviral plazmid inşa </li><li> 2 x 5 mg ambalaj inşa (EcoPak) </li><li> 2 x 62 ml CaCl </li><li> 1 ml steril GKD 2 O ile tamamlamak Not: retroviral plazmid faiz ve bir işaretleyici onkogen kodlar, EcoPak gag-pol-env kodlar. 293T hücreleri ile birlikte, bir fare hücreleri için spesifik ecotropic retrovirüs (RV), ama insan hücreleri için enfektif üretecektir. Klorokin ve CaCl 2 transfeksiyon verimliliği artırmak için gösterilmiştir. </li></ol></li><li> 2x sterileHBS 1ml ise tüp hafifçe karıştırın, karışımın açılan akıllıca ekleyin. </li><li> Hemen hafifçe 2 tabak, her biri 1 ml, bu çözümü eklemek damla bırak. </li><li> 7 ila 11 saat için kuvöz koyun. </li><li> Akşam (7 ila 11 saat sonra), yavaşça orta kaldırmak ve çok yavaşça 5 ml taze 293T orta ile değiştirin. , Ertesi gün öğlene kadar kuvöz koyun. Not: Bu adım, hücrelerin klorokin çıkarmak için ihtiyacınız olan önemlidir. Uzun süre devam ederse, klorokin toksiktir. Plakalarda çözüm transfeksiyon karışımı çok ince bir toz gibi yağış nedeniyle, bulanık görünüyor. Not: klorokin duyarlı hücreler olan ve plakası kolayca ayırmak gibi, aşırı yumuşaklığı ile tüm medya değişiklikleri yapmak önemlidir. </li><li> Ertesi gün, 18 ila 24 saat. Öğleyin) virüsü (almadan önce, hafifçe orta kaldırmak ve çok nazikçe 3 ml taze 293T orta ile değiştirin. Inkübatör O / N değiştirin. </li><li> Üçüncü sabah (18 ila 24 saat sonrası), yavaşça, 18 G iğne ile donatılmış 10 ml şırınga ile orta formu plakaları emmek. Bu süpernatant retrovirüs içerir. </li><li> ,, 45 mm şırınga filtre iğne, şırınga iyi çözüm karıştırmak için birkaç kez ters filtre ile süpernatant geçmek ve cryotubes içinde kısım Not: Alternatif olarak, 5 0 ml konik tüp içine virüs, havuz aynı virüs tüm süpernatantlar 3 + plaka yapıyoruz. Cryotubes retrovirüs içeren kısım süpernatantlar filtreleyerek, sonra iyice karıştırın. </li><li> Virüs dolu süpernatantlar içeren tüpler -80 ° C&#39;ye kadar içinde tutulur. </li></ol>

<ol>
	<li>DuBridge, R. B., Tang, P., Hsia, H. C., Leong, P. M., Miller, J. H., Calos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mutation+in+human+cells+by+using+an+Epstein-Barr+virus+shuttle+system.">Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.</a> Mol Cell Biol. 7, 379-387 (1987).</li><li>Cherry, S. R., Biniszkiewicz, D., van Parijs, L., Baltimore, D., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+expression+in+embryonic+stem+cells+and+hematopoietic+stem+cells.">Retroviral expression in embryonic stem cells and hematopoietic stem cells.</a> Mol Cell Biol. 20, 7419-7426 (2000).</li><li>Finer, M. H., Dull, T. J., Qin, L., Farson, D., Roberts, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=kat:+A+high-efficiency+retroviral+transduction+system+for+primary+human+T+lymphocytes.">kat: A high-efficiency retroviral transduction system for primary human T lymphocytes.</a> Blood. 83, 43-50 (1994).</li><li>Naviaux, R. K., Costanzi, E., Haas, M., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pCL+vector+system:+rapid+production+of+helper-free,+high-titer,+recombinant+retroviruses.">The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses.</a> J Virol. 70, 5701-5705 (1996).</li><li>Pear, W. S., Miller, J. P., Xu, L., Pui, J. C., Soffer, B., Quackenbush, R. C., Pendergast, A. M., Bronson, R., Aster, J. C., Scott, M. L., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+induction+of+a+chronic+myelogenous+leukemia-like+myeloproliferative+disease+in+mice+receiving+P210+bcr/abl-transduced+bone+marrow.">Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow.</a> Blood. 92, 3780-3792 (1998).</li><li>Pear, W. S., Nolan, G. P., Scott, M. L., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+high-titer+helper-free+retroviruses+by+transient+transfection.">Production of high-titer helper-free retroviruses by transient transfection.</a> Proc Natl Acad Sci USA. 90, 8392-8396 (1993).</li><li>Stocking, C., Kollek, R., Bergholz, U., Ostertag, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+terminal+repeat+sequences+impart+hematopoietic+transformation+properties+to+the+myeloproliferative+sarcoma+virus.">Long terminal repeat sequences impart hematopoietic transformation properties to the myeloproliferative sarcoma virus.</a> Proc Natl Acad Sci USA. 82, 5746-5750 (1985).</li><li>Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+chronic+myeloid+leukemia.">Models of chronic myeloid leukemia.</a> Curr Oncol Rep. 3, 228-237 (2001).</li><li>Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+pathogenesis+of+BCR-ABL++leukemia+in+mice.">Studying the pathogenesis of BCR-ABL+ leukemia in mice.</a> Oncogene. 21, 8643-8651 (2002).</li></ol>

Dört kritik noktaları iyi bir virüs stokunun başarısını temin edecektir: <ol><li> 293T üreten hücrelerin bir yoğunluk ulaşmak olduğunu, bu yüzden, çok düzenli olarak bölünmüş olan, yani çok sağlıklı olmak için büyümüş asla ve 6 cm doku kültürü plaka başına 3.5-5 milyon optimize yoğunlukta kaplama transfeksiyon sabahı plaka% 80. </li><li> Hücre lysozomes stabilize ve çekirdeği ulaşır DNA kesir artırarak, 5-kat klorokin eklenmesi yaklaşık 3 transfeksiyon verimliliği ve daha sonraki bir virüs titresi geliştirir. Sodyum butirat (başka lizozom sabitleyici) de bu amaçla kullanılmıştır. Klorokin değişen orta 7-11 saat bu durumda, ihmal edilebilir. Bu noktada post-transfeksiyon gerekli değildir. </li><li> DNA kalitesi çok önemlidir. Vektör ve ambalaj plazmid DNA&#39;lar hem de arıtma tercih edilen yöntem iki kez CSCL etidyum bromür batmaz yoğunluğu santrifüj arındırılır. Biz en az 1 mg / ml DNA konsantrasyonu ve OD 260/280 oranı 1,75-1,90 arasında olmalıdır. Qiagen-saflaştırılmış DNA yazarların elinde, sonuçlar iyi değildir, her ne kadar çalışacaktır. DNA çözümleri kombine hacimli kalsiyum fosfat çökelti Tris ve EDTA (TE) olumsuz etkilerini önlemek için (&lt;50 ul toplam son ml&#39;de) küçük olması önemlidir. </li><li> Ambalaj inşa retroviral vektör ve host aralığı seçimi önemlidir. Myeloproliferatif sarkomu virüsü dayalı bir vektör fare hematopoietik kök hücreler istikrarlı bir ifade için, tercih edilir. Ortak bir vektör omurgası MPSV uzun bir terminali Tekrar dizisi, bir onkogen tanıtımı için bir çok klonlama sitesi ve gelişmiş yeşil flüoresan protein (eGFP) gen ile bağlantılı bir alt-iç ribozom giriş yeri içeren, MIG RI. Fare HSC, virüs transdüksiyon için ecotropic konak aralığı en iyi, ancak hisse senetleri fizyolojik sıcaklığında kararsız ve santrifüj konsantre olamaz. </li></ol> Bazı kontrol noktaları: İsterseniz, bir kez hasat, 293 hücreleri retroviral vektör (örneğin, TK) tarafından kodlanan proteinlerin ekspresyonu kontrol etmek için immunoblotting protein lizatları yapmak için kullanılan olabilir. Ayrıca biz diğer plakaları süpernatant toplarken transfeksiyon efficinecy beta-gal testi ile kontrol etmek için ayrı tabak transfeksiyon zaman (ambalaj vektör gerekli) plazmid bir LacZ kodlama kontrolü kullanabilirsiniz. Not: Bu virüs yapmak için kullanılan plazmid kalitesini hücreleri ve reaktifler test, ama olmaz. Herhangi bir virüs kullanmadan önce, titresi tespit edilmesi gerekmektedir. Bu, aynı deneyi farklı virüs stokları, titreleri maç ve sipariş hematopoietik kök hücrelerin etkili transdüksiyon sağlamak için kritik öneme sahiptir.

Transfeksiyon kullanılan tüm reaktifler steril filtre edilir (CaCl 2, 2x HBS, miliQ H 2 O) ve steril tutulan gerekir.

production of replication-defective retrovirus by transient transfection of 293t cells

Bcr-Abl veya büyüme faktörü reseptör-kaynaklı onkogenler (ZNF198 FGFR1, Bcr-PDGFRα, vb) gibi onkogenler tarafından uyarılan laboratuar çalışmaları insan myeloproliferatif hastalıklar. Biz fare modelinde daha önce ilgi onkogen ifade bir retrovirüs ile enfekte kemik iliği hücrelerinin nakli, insan gibi bir hastalık modeli ve çalışma edebiliyoruz. Çoğaltma arızalı retrovirüs bir insan onkogen kodlama ve bir işaretleyici (GFP, RFP, antibiyotik direnç geni, vb.) 293T hücreleri, adenovirüs E1A gen ürünü tarafından dönüştürülmüş bir insan böbrek epitel hücre hattı kullanarak geçici bir transfeksiyon protokol tarafından üretilir. Kolayca ulaşılabilir% 50-80 transfeksiyon verimliliği kadar 293 hücreler, kalsiyum fosfat (Capo 4) yüksek transfectable olağandışı bir özelliği var . Burada, ilgi onkogen ve ambalaj Bu durumda kat veya PCL, yapıları tek genom EcoPak plazmid gibi gag-pol-env ambalaj fonksiyonları, ifade eden bir plazmid ifade retroviral vektör ile 293 hücreleri transfect ortak. Ilk transfeksiyon daha klorokin kullanımı artırıldı. Ecotropic virüs Hisse Senetleri, kültür süpernatant 48 saat olarak toplanır. post-transfeksiyon, C ° -80 saklanan ve dönüşüm görünümünde ve in vitro çalışmalardan sonra fare modeli nakli için kullanılabilir hücre hatları, ya da, fare kemik iliği hücreleri gibi birincil hücreleri, enfeksiyon için kullanılır. .

Watch this Scientific Journal Video about Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells at JoVE.com

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Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Bu teknik, bir insan onkogen kodlama çoğaltma kusurlu retroviral stokları hazırlamak için etkin bir şekilde gösteriyor ve daha sonra fare modeli myeloproliferatif hastalığı indüksiyonu için kullanılır.

293T hücreleri geçici Transfeksiyon Çoğaltma-Arızalı Retrovirus Üretimi

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

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19.5K Görüntüleme.  Tufts University. Bu teknik, bir insan onkogen kodlama çoğaltma kusurlu retroviral stokları hazırlamak için etkin bir şekilde gösteriyor ve daha sonra fare modeli myeloproliferatif hastalığı indüksiyonu için kullanılır.

Video: Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Gene-gun Transfection of Hippocampal Neurons

Hi, I'm Kma. I work in Case Lab at Brown University Clean Slicer stage with 70%ethanol. Place plate insert into six.Well plate with medium and incubate at 37 degree whole brain with tweezers between midbrain and four brainin. Isolate hippocampus on one side, isolate hippocampus from the second side. Place hippocampi onto A PBS drop.Aspirate out the PBS make sagittal sections using the tissue slicer. Repeat transfer slicers onto a perish by squatting with PBS wash sections by adding and aspirating PBS and medium gently. Separate sections with tweezers.Wash sections with PBS and media. Again, transfer sections onto the plate and insert. Aspirate out the media in sections using tweezers.Attach gene gun hose to the helium cylinder. Attach barrel to gene gun. Fill the magazine with bullets.Insert magazine into the Jing gun. Adjust the pressure on the helium tank shoot. EGFP coated beads onto the sections.And now.

Gen tabancası Transfeksiyon Hipokampal Nöronlar

Biyoloji

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

Rekombinant mCD1d Böcek Hücrelerde Protein Üretimi İçin Bir Yöntem.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Transgenik, üretimi Xenopus laevis Kısıtlama Enzim aracılığı ile Entegrasyon ve Nükleer Nakli

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

İfade ve HEK-293T Hücreler Rekombinant Gerilim Gated İyon Kanalları Elektrofizyolojik Kayıt için optimize edilmiş Transfeksiyon Strateji

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HeLa Hücre Transfeksiyon Ardından Gaussia Lusiferaz mRNA in vitro transkripsiyon ve Kapatma yılında

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Protein Transfection of Mouse Lung

Increasing protein expression enables researchers to better understand the functional role of that protein in regulating key biological processes1. In the lung, this has been achieved typically through genetic approaches that utilize transgenic mice2,3 or viral or non-viral vectors that elevate protein levels via increased gene expression4. Transgenic mice are costly and time-consuming to generate and the random insertion of a transgene or chronic gene expression can alter normal lung development and thus limit the utility of the model5. While conditional transgenics avert problems associated with chronic gene expression6, the reverse tetracycline-controlled transactivator (rtTA) mice, which are used to generate conditional expression, develop spontaneous air space enlargement7. As with transgenics, the use of viral and non-viral vectors is expensive8 and can provoke dose-dependent inflammatory responses that confound results9 and hinder expression10. Moreover, the efficacy of repeated doses are limited by enhanced immune responses to the vector11,12. Researchers are developing adeno-associated viral (AAV) vectors that provoke less inflammation and have longer expression within the lung13.
Using &beta;-galactosidase, we present a method for rapidly and effectively increasing protein expression within the lung using a direct protein transfection technique. This protocol mixes a fixed amount of purified protein with 20 &mu;l of a lipid-based transfection reagent (Pro-Ject, Pierce Bio) to allow penetration into the lung tissue itself. The liposomal protein mixture is then injected into the lungs of the mice via the trachea using a microsprayer (Penn Century, Philadelphia, PA). The microsprayer generates a fine plume of liquid aerosol throughout the lungs. Using the technique we have demonstrated uniform deposition of the injected protein throughout the airways and the alveoli of mice14. The lipid transfection technique allows the use of a small amount of protein to achieve effect. This limits the inflammatory response that otherwise would be provoked by high protein administration. Indeed, using this technique we published that we were able to significantly increase PP2A activity in the lung without affecting lung lavage cellularity15. Lung lavage cellularity taken 24 hr after challenge was comparable to controls (27&plusmn;4 control vs. 31&plusmn;5 albumin transfected; N=6 per group). Moreover, it increases protein levels without inducing lung developmental changes or architectural changes that can occur in transgenic models. However, the need for repeated administrations may make this technique less favorable for studies examining the effects of long-term increases in protein expression. This would be particularly true for proteins with short half-lives.

Transgenic mice or viral vectors have been used to increase protein expression within the lung. However, these techniques are time-consuming, technically challenging and have off-target effects that can confound results. Our protein transfection protocol uses a lipid based transfection reagent and an ultrafine microsprayer to uniformly deliver active protein to lung cells.

Fare Akciğer Protein Transfeksiyon

Increasing protein expression enables researchers to  better understand the functional role of that protein in regulating key  biological processes1. In ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Optimizasyon ve Kullanımı<em&gt; Agrobacterium</emIn&gt; aracılı Geçici Protein Üretimi<em&gt; Nicotiana</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

Farelerde Hidrodinamik Gen Taşıyıcı Proteinlerin Geçici İfadesi

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

Haploit Zebra balığı Embriyolar üretimi ile<em&gt; In Vitro</em&gt; Gübreleme

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

293T hücreleri geçici Transfeksiyon Çoğaltma-Arızalı Retrovirus Üretimi

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