<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ערב קודם, השינוי התאים צלחת 293T ב 3-5x10 6 תאים / 6 cultureplate רקמות ס&quot;מ. הערה: אנו משתמשים 293T תאים על מנת להקל שלהם transfection ותאי efficacyas וירוס לייצר. תאים אלו חסרים האריזה וירוס אלא עוזר פלסמיד הוא הציג. </li><li style=";text-align:right;direction:rtl"> בבוקר הראשון, בעדינות להסיר ולהחליף בינוני עם 4 מ&quot;ל 293T תא התרופה המכילה 25 chloroquine מ&quot;מ. מכניסים חממה שעות 1. הערה: הצלחת צריך להיות בערך 80% ומחוברות. </li><li style=";text-align:right;direction:rtl"> בינתיים, להכין וירוס קבלת תערובת עבור 2 צלחות בכל פעם, ב 5 צינורות מ&quot;ל: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 2 x 10 מ&quot;ג לבנות פלסמיד retroviral </li><li style=";text-align:right;direction:rtl"> 2 x 5 מ&quot;ג האריזה לבנות (EcoPak), </li><li style=";text-align:right;direction:rtl"> 2 x 62 מ&quot;ל CaCl </li><li style=";text-align:right;direction:rtl"> להשלים עם 1 מ&quot;ל סטרילי DDH 2 O הערה: בעוד retroviral פלסמיד המקודד את oncogene עניין סמן, EcoPak מקודד איסור הפרסום-pol-env. יחד עם תאים 293T, הם יוכלו לייצר רטרווירוס ecotropic (RV) ספציפית בתאי עכבר, אך לא זיהומיות עבור תאים אנושיים. שני chloroquine, CaCl 2 הוכחו להגביר את היעילות transfection. </li></ol></li><li style=";text-align:right;direction:rtl"> הוסף 1ml של sterileHBS 2x ירידה מבחינת לתערובת, תוך vortexing בעדינות את הצינורית. </li><li style=";text-align:right;direction:rtl"> מיד להוסיף את פתרון 2 צלחות, 1 מ&quot;ל כל אחת, בעדינות, טיפה אחר טיפה. </li><li style=";text-align:right;direction:rtl"> שים באינקובטור במשך 7 עד 11 שעות. </li><li style=";text-align:right;direction:rtl"> בשעות הערב (7-11 שעות. מאוחר יותר), להסיר בעדינות בינוני מאוד בעדינות להחליף עם בינוני 5 מ&quot;ל 293T טריים. מכניסים החממה עד הצהריים, למחרת. הערה: שלב זה הוא חשוב, אתה צריך לקחת את chloroquine מן התאים. אם כל הזמן לפרקי זמן, chloroquine רעיל. הפתרון של הצלחות נראה מטושטש, בשל משקעים מצוין כמו אבק, של התערובת transfection. הערה: חשוב לעשות את כל השינויים התקשורת בעדינות קיצוניים, כמו התאים להיות רגיש על ידי chloroquine, והוא יכול בקלות לנתק מן הצלחת. </li><li style=";text-align:right;direction:rtl"> למחרת, 18-24 שעות. לפני שלקח את וירוס (בצהריים), להסיר בעדינות בינוני מאוד בעדינות להחליף עם המדיום מ&quot;ל 3 293T טריים. החלף באינקובטור O / נ </li><li style=";text-align:right;direction:rtl"> בבוקר השלישי (18-24 שעות. מאוחר יותר), בעדינות למצוץ את הטופס המדיום צלחות עם מזרק 10 מ&quot;ל מצויד במחט G 18. Supernatant זה מכיל את רטרווירוס. </li><li style=";text-align:right;direction:rtl"> שנה את המחט לסנן מזרק 45 מ&quot;מ, להפוך מזרק מספר פעמים כדי לערבב את פתרון טוב, לעבור דרך supernatant סינון aliquot ב cryotubes הערה: לחילופין, אם אתה עושה 3 + צלחות של הנגיף, בריכת כל supernatants של הנגיף אותו לתוך צינור 5 מ&quot;ל 0 חרוטי. מערבבים היטב, אז supernatants aliquot המכיל את הרטרווירוס cryotubes ידי סינון. </li><li style=";text-align:right;direction:rtl"> צינורות המכילים את הנגיף מלא supernatants נשמרים -80 ° C עד בשימוש. </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>

ארבע נקודות קריטיות יבטיח את ההצלחה של מניות ויראלי טוב: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> התאים לייצר 293T צריך להיות מאוד בריא, כלומר הם היו מפוצלים לפי לוח זמנים קבוע מאוד, מעולם לא היו מגודלים, והם מצופה בצפיפות של 3.5-5000000 מותאם לכל צלחת 6 ס&quot;מ תרבות רקמות, כד...

כל ריאגנטים בשימוש transfection חייב להיות סטרילי, מסוננים (CaCl 2, 2x בהרוורד, miliQ H 2 O) והמשיך סטרילית.

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

מחקרים במעבדה שלנו מחלות myeloproliferative האדם הנגרמת על ידי אונקוגנים כגון Bcr-Abl או גורם הגדילה לקולטן הנגזרות אונקוגנים (ZNF198-FGFR1, Bcr-PDGFRα וכו &#39;). אנו מסוגלים מודל מחקר מחלות אנושיות, כמו במודל העכבר שלנו, על ידי השתלת תאים במח העצם נגוע בעבר עם רטרווירוס ביטוי oncogene של עניין. שכפול-פגום רטרווירוס קידוד oncogene האדם סמן (GFP, RFP, גן עמידות לאנטיביוטיקה, וכו &#39;) מופק על ידי פרוטוקול transfection חולף באמצעות תאים 293T, קו אנושי כליות תא אפיתל ממיר את המוצר adenovirus גן E1A. 293 תאים יש את המאפיין יוצא דופן להיות transfectable מאוד על ידי סידן זרחתי (Capo 4), עם יעילות של עד 50-80% transfection להשגה בקלות. כאן, אנו שיתוף transfect 293 תאים עם וקטור retroviral ביטוי oncogene עניין פלסמיד המבטא את איסור הפרסום-pol-env פונקציות אריזה, כגון הגנום יחיד אריזה בונה קאט או PCL, במקרה זה EcoPak פלסמיד. Transfection הראשונית היא שיפור נוסף על ידי שימוש chloroquine. מניות של הנגיף ecotropic, שנאספו supernatant תרבות 48 שעות. פוסט transfection, ניתן לאחסן -80 ° C ו המשמש זיהום של קווי תאים לאור השינוי במבחנה, או תאים ראשוניים כגון עצם לתאי מוח העכבר, כי לאחר מכן ניתן להשתמש להשתלה במודל העכבר שלנו .

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

טכניקה זו ממחישה דרך יעילה כדי להכין את השכפול פגום מניות retroviral קידוד oncogene אדם, לאחר מכן נעשה שימוש לזירוז המחלה myeloproliferative במודל עכבר.

הפקה של רטרווירוס שכפול פגומים-by transfection חלוף של תאים 293T

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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19.4K Views.  Tufts University. טכניקה זו ממחישה דרך יעילה כדי להכין את השכפול פגום מניות retroviral קידוד oncogene אדם, לאחר מכן נעשה שימוש לזירוז המחלה myeloproliferative במודל עכבר.

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

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).

Transfection אסטרטגיה אופטימיזציה עבור הביטוי הקלטה של ​​אלקטרו Voltage-מגודרת ערוצי רקומביננטי יון ב HEK-293T תאים

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

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.

אופטימיזציה וניצול<em&gt; Agrobacterium</emייצור חלבון חלוף&gt; בתיווך ב<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.

ביטוי חולף של חלבונים על ידי הידרודינמית ג&#39;ין משלוח בעכברים

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.

במעקב 4 ממדי Vivo של תאי גזע ואבות היווצרות דם במבוגרי עכבר calvarial מח עצם

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.

ייצור של הפלואידים עוברי דג הזברה על ידי<em&gt; במבחנה</em&gt; הפריה

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

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

ביטוי תשואה גבוהה של חלבונים אנושיים רקומביננטי עם transfection חולף של תאי HEK293 בהשעיה

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

ביטוי חולף ואת סלולר לוקליזציה של חלבונים רקומביננטי תאים בתרבית חרקים

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

חקר ביטוי חלבונים במהלך התפתחות עדשת עוף

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of similarity with the human lens. RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells, which serves as a powerful tool to study the in situ expression and function of wild-type and mutant proteins during lens development by microinjection into the empty lumen of lens vesicle at early developmental stages, restricting its action to surrounding proliferating lens cells. Compared to other approaches, such as transgenic models and ex vivo cultures, the use of an RCAS(A) replication-competent avian retrovirus provides a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Specifically, targeted gene transfer can be confined to proliferative lens fiber cells without the need for tissue-specific promoters. In this article, we will briefly overview the steps needed for recombinant retrovirus RCAS(A) preparation, provide a detailed, comprehensive overview of the microinjection procedure, and provide sample results of the technique.

מחבר זרקור: חקירת התפתחות ותפקוד עדשה באמצעות מיקרו-הזרקה של רטרו-וירוס RCAS(A) בעדשת עוף עוברית 

This protocol paper describes the methodology of embryonic chicken lens microinjection of an RCAS(A) retrovirus as a tool for studying in situ function and expression of proteins during lens development.

מיקרו-הזרקה של רטרו-וירוס רקומביננטי RCAS(A) לעדשת עוף עוברית

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of ...