המחברים מבקשים להודות קארן לורנס על עזרתה בבניית קטע המתאר של החדר electroporation.

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אין ניגודי אינטרסים הכריז.

Electroporation Explant הוא אמצעי פשוט של לכימות cis-הרגולציה פעילות ברשתית עכבר מתפתחות. בהשוואה לניתוח cis-הרגולציה transgenesis באמצעות העכבר, electroporation הוא הרבה יותר זול, המחייב גורים עכבר היילוד בלבד, DNA, כלי לנתח, electroporation ו / תרבות ציוד רקמות. הוא גם הרבה פחות זמן רב: הניסוי אחד דורש רק כמה שעות של זמן הכנה, תקופה תרבות של כשמונה ימים, ולאחר כמה שעות בסוף הניסוי הדמיה וניתוח נתונים. Electroporation Explant היא גם מעולה לניתוח תרבות מבוססי cis-הרגולציה התא מאז רקמת הרשתית בפועל הוא מנוצל. הרשתית מתפתחת בדרך כלל די explant תרבות היא יוצרת שלוש שכבות נפרדות הסלולר (שכבת הגרעין החיצונית, שכבת הגרעין הפנימי, התא שכבת הגנגליון), למרות photoreceptors להיכשל לפרט מגזרים החיצונית. יתרון נוסף הוא כי electroporation explant ניתנת לשחזור מאוד. בונה אותו electroporated לתוך הרשתית שונים, אפילו בימים שונים, בדרך כלל התוצאות באותן הרמות ביטוי יחסית. יתר על כן, מאז פלסמידים electroporated נחשבים שמרו episomally בגרעין ולא ישולבו כרומוזומים, הם אינם מופיעים להיות כפוף לאתר אותן השפעות האינטגרציה כי לבלבל cis-הרגולציה ניתוח שבוצע העכברים הטרנסגניים. Electroporation Explant יש מספר מגבלות. ראשית, התאים רק כי הם עדיין מחזור התא יכול להיות יעיל transduced ידי electroporation 15. ב P0, מוטות ושאר מאוחר יותר, יליד סוגי תאים ברשתית (תאים דו קוטבית, תאים amacrine, גליה M ller) הם אוכלוסיות תאים העיקרי ממוקד בשיטה זו. Electroporation של photoreceptors חרוט על ידי electroporation P0 דווחה 16 אך היעילות נראה נמוך. המגבלה השנייה היא תרבות explant מעבר שתי תוצאות שבועות מום הדרגתי של הרשתית ולכן אינו מומלץ. אם כימות היזם נדרש על timepoints מאוחר, לעומת זאת, ב electroporation vivo מאי 9 להתבצע, ואחריו לנתיחה רשתית ב timepoint הרצוי שטוח הרכבה של הרשתית גזור, וכימות, כמתואר בסעיף 9. המגבלה השלישית היא assay זה הוא רק בינוני תפוקה גבוהה. בניגוד לתרבות המבוססת על מבחני התא יכול לבדוק את מאות המבנים בניסוי אחד, את הטכניקה המתוארת פרוטוקול זה דורש מינימום של הרשתית עכבר אחד שלם לכל לבנות. לפיכך, רק תריסר בונה כמה סביר electroporated ביום. אזהרה אחת נוספת לגבי כימות של הפעילות באמצעות מקדם את הגישה הנוכחית היא כי קיים פוטנציאל &quot;לדמם דרך&quot; של הקרינה DsRed לתוך התעלה GFP, במיוחד כאשר assaying היזמים חזקה מאוד. הסיבה לכך היא כי ספקטרום פליטה של ​​DsRed חופף חלקית את זה של ה-GFP. כדי לעקוף בעיה זו, מסנני פליטה מותאם אמור לשמש כי למזער את החפיפה בין ספקטרלי DsRed ו-GFP. כאשר כגון מערכות סינון אופטימיזציה אינם זמינים, עוד פתרון אפשרי יהיה להשתמש חלבון כחול מוזז ניאון (למשל, BFP או CFP) במקום ה-GFP.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> שם מגיב </th><th> חברה </th><th> מספר קטלוגי </th><th> הערות (אופציונלי) </th></tr><tr><td> צלחת electroporation, Microslide 453 </td><td> BTX הרווארד Apparatus </td><td> 45-0105 </td><td> ראה סעיף 1 בפרוטוקול לשינויים </td></tr><tr><td> 100% גומי סיליקון מלט אקווריום </td><td> פרפקטו ייצור </td><td></td><td></td></tr><tr><td> Microtube פלסטיק מתלה </td><td> פישר סיינטיפיק </td><td> 05-541 </td><td> רק להתמודד עם מתלה ישמש </td></tr><tr><td> כלי Dremel </td><td></td><td></td><td> לחיתוך את הידית מתלה צינור פלסטיק </td></tr><tr><td> DMEM </td><td> Gibco / Invitrogen </td><td> 11965 </td><td></td></tr><tr><td> F12 </td><td> Gibco / Invitrogen </td><td> 11765 </td><td></td></tr><tr><td> L-Glu/pen/strep </td><td> Gibco / Invitrogen </td><td> 10378-016 </td><td> 100x ריכוז </td></tr><tr><td> אינסולין </td><td> סיגמא אולדריץ </td><td> I-6634 </td><td> עבור המניות 1000x, resuspend ב 5mg/ml ב HCl 5mm ולסנן-לעקר </td></tr><tr><td> FBS </td><td> Gibco / Invitrogen </td><td> 26140-079 </td><td></td></tr><tr><td> ECM 830 כיכר גל electroporator </td><td> BTX הרווארד Apparatus </td><td></td><td></td></tr><tr><td> Nuclepore מסננים </td><td> Whatman </td><td> 110606 </td><td> 25mm, 0.2μm </td></tr><tr><td> רקמות תרבות חממה </td><td></td><td></td><td> 37 מעלות צלזיוס, 5% CO 2 </td></tr><tr><td> זכוכית coverslips # 1.5 </td><td> פישר סיינטיפיק </td><td> 12-544E </td><td> עבה 0.16mm </td></tr><tr><td> תרכובת מיקרוסקופ פלורסנט מצויד במצלמה </td><td></td><td></td><td> המצלמה צריכה להיות חד גוני (למשל, ORCA-ER מצלמה ידי Hamamatsu) </td></tr><tr><td> EGFP / להגדיר מסנן DsRed עבור מיקרוסקופ מתחם </td><td> Chroma טכנולוגיה קורפ </td><td> 86007 </td><td> זה bleedthrough להגדיר מסנן ממזער chanenels בין אדום וירוק </td></tr><tr><td> ImageJ תוכנה </td><td> NIH </td><td></td><td> <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> </td></tr></tbody></table>

quantifying the activity of cis-regulatory elements in the mouse retina by explant electroporation

גורמי שעתוק בתוך רשתות גנים הסלולר לשלוט דפוס spatiotemporal ורמות הביטוי של גנים היעד שלהם באמצעות קשירה cis-הרגולציה אלמנטים (צרס), קצר (~ 300-600 נ&quot;ב) מותח של הדנ&quot;א הגנומי אשר יכול לשקר במעלה הזרם, במורד הזרם, או בתוך אינטרונים הגנים הם שולטים. צרס (כלומר, משפרי / היזמים) בדרך כלל מורכב מרובים אתרי הקישור אשכולות הן activators ו repressors תעתיק 1-3. הם משמשים אינטגרטורים הלוגי של קלט תעתיק מתן פלט יחידתי בצורה של פעילות האמרגן מדויק spatiotemporally ומדויק כמותית. רוב המחקרים של יונקים cis-תקנה עד כה הסתמכה על transgenesis העכבר כאמצעי assaying משפר את הפונקציה של צרס 4-5. טכניקה זו זמן רב, יקר, על חשבון תופעות באתר הכנסת, בעיקר לא כמותי. מצד שני, מבחני כמותיים לתפקוד Cre יונקים פותחו מערכות תרבות רקמות (למשל, מבחני בלוציפראז כפול), אבל ב vivo הרלוונטיות של תוצאות אלו היא לעיתים קרובות לא ברור. Electroporation מציע אלטרנטיבה מצוינת transgenesis העכבר המסורתי בכך שהוא מאפשר גם spatiotemporal הערכה כמותית של פעילות cis-הרגולציה רקמה חיה יונקים. טכניקה זו הייתה שימושית במיוחד בניתוח של רגולציה-cis במערכת העצבים המרכזית, בעיקר בקליפת המוח ואת רשתית 6-8. בעוד electroporation עכבר הרשתית, הן in vivo לשעבר vivo, פותחה ותיאר בהרחבה על ידי מאטסודה ו Cepko 6-7,9, פיתחנו לאחרונה גישה פשוטה לכמת את הפעילות של photoreceptor ספציפי צרס ב electroporated עכבר הרשתיות 10. בהתחשב בכך את כמות ה-DNA כי הוא הציג לתוך הרשתית על ידי electroporation יכול להשתנות מניסוי לניסוי, יש צורך לכלול &quot;טעינה מלאה&quot; שותף electroporated בניסויים בכלל. במובן זה, הטכניקה דומה מאוד assay בלוציפראז כפול המשמש לכמת פעילות הפרומוטר בתאים בתרבית. כאשר assaying photoreceptor cis-הרגולציה פעילות, electroporation מתבצעת לרוב בעכברים היילוד (יום הלידה 0, P0) המהווה את זמן הייצור מוט שיא 11-12. ברגע סוגי תאים ברשתית להיות שלאחר mitotic, electroporation הוא הרבה פחות יעיל. לנוכח שיעור גבוה של לידת מוט בעכברים הילוד לבין העובדה מוטות מהוות יותר מ 70% של תאים ברשתית עכבר מבוגר, הרוב המכריע של התאים electroporated ב P0 הן מוטות. מסיבה זו, photoreceptors מוט הם סוג הקלה תא רשתית ללמוד דרך electroporation. הטכניקה שאנו מתארים כאן הוא שימושי בעיקר עבור לכימות פעילות של צרס photoreceptor.

Watch this Scientific Journal Video about Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation at JoVE.com

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

פרוטוקול זה מתאר דרך פשוטה וזולה לכמת את הפעילות של cis-הרגולציה אלמנטים (כלומר, משפר / היזמים) לחיות הרשתיות העכבר דרך electroporation explant. הכנת ה-DNA, דיסקציה רשתית, electroporation, תרבות explant רשתית, שלאחר קיבוע ניתוח וכימות מתוארים.

כימות של פעילות Cis-Regulatory אלמנטים ברשתית עכבר ידי electroporation Explant

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

quantifying-activity-cis-regulatory-elements-mouse-retina-explant

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

14.4K Views.  Washington University School of Medicine. פרוטוקול זה מתאר דרך פשוטה וזולה לכמת את הפעילות של cis-הרגולציה אלמנטים (כלומר, משפר / היזמים) לחיות הרשתיות העכבר דרך electroporation explant. הכנת ה-DNA, דיסקציה רשתית, electroporation, תרבות explant רשתית, שלאחר קיבוע ניתוח וכימות מתוארים.

Video: Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Transfection של תאים עכבר רשתית גנגליון על ידי In vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

ב electroporation vivo של הרשתית עכבר פיתוח

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

ין העברת לאוזן הפנימית על ידי פיתוח מאוס<em&gt; In vivo</em&gt; Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

מניפולציה גנטית של העכבר באמצעות פיתוח היפותלמוס<em&gt; ברחם</em&gt; Electroporation

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

הערכה של כלי דם התחדשות במערכת העצבים המרכזית באמצעות רשתית העכבר

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

פעילות telomerase באזורים שונים של מוח עכבר: הטלומרז לא רדיואקטיבי חזור הגברה פרוטוקול Assay (TRAP)

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

ההדמיה Ca<sup&gt; 2 +</sup&gt; Dynamics בסופי קון קולטי האור אקסון של רשתית העכבר

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

In vivo ג&#39;ין העברה בתאי שוואן בעצב מכרסמים Sciatic ידי Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

התחדשות האקסון בתרבית של החוקר: In Vivo אלקטרופורציה של העכבר למבוגרים העזוב שורש גנגליון

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

לכימות ההתפלגות הטרוגנית של חלבון סינפטיים במוח העכבר בעזרת Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...