Создание генной пушки пулями (GOLD) <ol><li> Сделать 100X ПВП (0.5mg/ml) в 100% этанола. </li><li> Взвесьте из 30 мг BioRad 1,6 золота Micron микроносителей и поместить его в трубку 1,5 мл Eppendorf. </li><li> Рассчитать количество плазмиды, необходимых для получения концентрации 0,5-3 мкг плазмиды / мг золота, и место плазмиды в трубке с золотым микроносителях. Комбинат несколько плазмид, если необходимо. </li><li> Довести объем плазмиды до 100 мкл дистиллированной воды. </li><li> Добавить 100 мкл 0,05 М спермидина в трубку. Вихревые трубки 30 секунд. </li><li> Vortex 1 минуты на полной скорости. Разрушать ультразвуком в течение 3-5 минут .. </li><li> Сухой соответствующей длины Tefzel труб в аппарате Biorad PDS-1000/He течение 15 минут. </li><li> После обработки ультразвуком, вихревые в течение 1-2 минут на полной скорости. Снизить скорость медленно, чтобы трубка будет открыт в то время как вортексе. </li><li> Добавить 100 мкл 1 М CaCl 2 медленно трубки капля в то время как мудрые вортексе. </li><li> Оставьте трубки при комнатной температуре в течение 10 минут. </li><li> Сделайте 5 микрон фильтр. <ol><li> Возьмите два 5 мл шприца без иглы. </li><li> Удалите поршень, и отделить черные шапки. Принимая маленькие ножницы, отрезали часть черной кепке, чтобы дать кольцо. Повторите с другой поршень с чистым два кольца. </li><li> Сэндвич 5 микрон (мелкие детали, Inc нейлоновый фильтр, ½ дюйма в диаметре) фильтр между двумя кольцами, и нажимаем бутерброд на кончике шприца. </li><li> Мотаться через шприц 50 мл коническую колбу. </li></ol></li><li> Через 10 минут, вихревые трубки в течение 30 секунд. </li><li> Центрифуга трубку в течение 30 секунд и сохранить гранул. </li><li> Добавить 1 мл 100% этанола для осаждения. Использование пипетки, чтобы разбить гранул, и вихрь 30 секунд. Спиновые 30 секунд и снять этанола. Повторить еще 2 раза. </li><li> Добавить 1 мл 100% этанола в гранулах (перерыв с наконечником). Место это в 5 микрон фильтр и дайте ему течь через под действием силы тяжести в 50 мл коническую трубку. Используйте больше этанола, сколько необходимо для промыть трубы или помочь с потоком до конца. </li><li> Спиновые 50мл трубки при 2000 оборотов в минуту в центрифуге в течение 5-10 минут. Удалить супернатант, за исключением гранул. </li><li> В зависимости от желаемой плотности микроносителях необходимо, добавьте 800μl-1,5 мл 100% этанола и соответствующих 8 мкл-15 мкл раствора ПВП. </li><li> Swirl и сразу же пойти в аппарате PDS-1000/He Biorad. </li><li> Biorad PDS-1000/He аппарата <ol><li> Отсоедините трубку, соединяющую бак N2 газа в трубке сушки </li><li> Выньте сушки Tefzel трубы от машины </li><li> Вставьте один конец в 50 мл коническую трубку, и присоединить шприц к другому концу трубы. Соси коричневого шлама в середине трубы. </li><li> Вставьте трубку обратно в машину. </li><li> Wait (без вращения) в течение 4 минут </li><li> После 4-х минут, отсасывать этанола медленно и осторожно, стараясь не нарушить микроносителях. </li><li> Поворот трубы на 1 минуту. </li><li> Через 1 минуту подключения трубки, соединяющей N 2 бензобак в сушильную трубку и пусть трубы высохнуть в течение 15 минут. </li></ol></li><li> Через 15 минут, разрезать трубы, чтобы пуля размер будет использоваться сразу или хранить при температуре 4 ° С в эксикаторе. Пули могут храниться до 3 месяцев. </li></ol> Подготовка генной пушки <ol><li> Место пули в картридже. Каждая часть сетчатки могут быть сняты с 1-3 пуль. </li><li> Вставьте картридж в генной пушки и прикрепить пистолет к гелия источник. Установить давление до 110 пси. </li></ol> Подготовка средств массовой информации <ol><li> Препарирование среда <table style="width: 229px; height: 157px;" border="0"><tbody><tr><td> 1 бутылка Эймс решение (Sigma, 8.8g) </td></tr><tr><td> 1,9 г натрия гидрокарбоната </td></tr><tr><td> 10 мл 1% пера / стрептококк / L-глютамин (1:100) (Gibco / Invitrogen) </td></tr><tr><td> 990 мл дистиллированной водой до 1 л </td></tr><tr><td> ------------------------------- </td></tr><tr><td> 1 литр </td></tr></tbody></table> </li><li> Фильтры рассечение среды с использованием 0,22 мкм фильтр. </li><li> Инкубационный среде (500 мл) <table style="width: 283px; height: 157px;" border="0"><tbody><tr><td> 485 мл с фильтром СМИ рассечение </td></tr><tr><td> 5 мл 1% N2 дополнения (Gibco / Invitrogen) </td></tr><tr><td> 10 мл 1% лошадиной сыворотки (Sigma) </td></tr><tr><td> 500 мкл фенола красного </td></tr><tr><td> -------------------------------------------------- - </td></tr><tr><td> 0,5 л </td></tr></tbody></table> </li><li> Фильтры среду инкубации использованием 0,22 мкм фильтр. </li><li> Bubble O 2 в среде рассечение в течение 15 минут перед началом протокола сетчатки уборки урожая. </li></ol> Подготовка инкубации камеры для сетчатки <ol><li> Под капотом, заполнить несколько 60 мм в глубину блюда (инкубационный камер, Nunc) с 25 мл инкубационной среды. Количество блюд зависит от числа отставкеInAs необходимости. </li><li> Место фильтр стоять в каждую глубокую тарелку. Только самые кончики стоять не должен быть погружен в воду. </li><li> Поместите все глубокие блюда в инкубаторе до сетчатки готовы. </li><li> Заполните два 100 мм чашки Петри и довести до вскрытия скамейке. </li></ol> Подготовка кролика сетчатки <ol><li> Жертва кролика в соответствии с установленными протоколами. Следующие не должна быть выполнена под капотом. </li><li> С угловыми ножницами, вставьте его за глаза в глазнице из кролика, резка оболочки и зрительного нерва, которые прикреплены к глазу. </li><li> Аккуратно выньте глаз и промойте его кислородом СМИ рассечение. </li><li> Место глаза (роговица вверх) в колодец глаз резки и закрыть крышкой. </li><li> Место лезвием горизонтально прямо под крышкой. В одном движении, рисовать лезвием поперек глаз, чтобы отрезать роговицы, в результате чего глаз чашку. </li><li> Использование щипцов, удалить сетчатку глаза от среза хорошо и поместить его на фильтровальную бумагу. </li><li> Удаление стекловидного тела и линзы, зажимая области около разорвала зрительного нерва и потянув назад на фильтровальную бумагу. Повторите несколько раз, пока почти все стекловидное удаляется и наглазник выглядит спущена. </li><li> Место глаз чашки в чашку Петри с рассечением СМИ, чтобы помыться. </li><li> Вырезать глаза чашу в желаемое количество штук (5 штук максимум). </li><li> Возьмите одну часть глаза чашку и поместить его под микроскопом. </li><li> Зажим склеры и сосудистой оболочки легко собираются на краю сетчатки использованием щипцов. С другой стороны, использование тонкой кистью, чтобы кисть правой между сосудистой оболочки и сетчатки. </li><li> Использование коротких, нежных движений, дразнить сетчатки с сосудистой оболочкой, пока не отрывается. </li><li> Вырезать 1 дюйм от стерильной пипетки передачи. Используйте это, чтобы подлизываться сетчатки и положите в чашку Петри в среде инкубации мыть. </li><li> Используя другой стерильной пипетки передачи с 1 дюйм отрезать, тщательно передачи сетчатки на 0,4 мкм Millicell фильтром (Millipore, Cat No: PICMORG50). </li><li> Используйте кисточку, чтобы ориентировать сетчатке стороны ганглиозных клеток вверх. </li><li> Свести из сетчатки кистью слегка по краям сетчатки. Свернуть трогательные слой ганглиозных клеток с помощью кисти как можно больше. </li><li> Удалите излишки среды на фильтр с передачей пипетки. </li><li> Использование щипцов, передачи фильтра на изменение suctioner. Нарисуйте на suctioner 2-3 раза, чтобы удалить все среде инкубации с фильтром. </li></ol> Торкрет Гена и инкубации сетчатки <ol><li> Под капотом, место каждого из сетчатки содержащих-фильтров на фильтр стоит в 60 мм в глубину блюда (инкубационный камеры). </li><li> Убедитесь, что среда находится в контакте с нижней фильтр и что среда не перекинуться стороны фильтра на сетчатку. Фильтр должен выступать в качестве интерфейса между сетчаткой и среды. </li><li> Съемка каждой части сетчатки с двумя пулями. Место генной пушки прямо над ткани, около 3-4 см прочь. </li><li> Поместите все камеры инкубации на шейкере в 35 - 37 ° C инкубатора. </li><li> Замените среду инкубации с каждым днем. Сетчатки может быть инкубировали в течение не более 6 дней. </li></ol>

<ol>
	<li>Landreth, . B. W., Agranoff, N. R., Smalheiser, S. M., Crain, M. B., Bornstein, R., Adler, P. J., Magistretti, A. G., Hyndman, W. J., Shoemaker, H., Rehm, T., Schafer, H., Betz, S. U., Kim, H., Takahashi, A. R., Caffe, A., Soderpalm, T., van Veen, H. C., Wong, S., Thompson, D. Y., Yu, S. J., Cringle, V. A., Alder, S. J., Taylor, G. M., Seigel, A. R., Caffe, P., Ahuja, B., Holmqvist, S., Azadi, J., Forsell, I., Holmqvist, J., Hatakeyama, R., Kageyama, S. S., Zhang, X. Y., Fu, C. J., Barnstable, S. W., Wang, X., Mu, W. J., Bowers, W. H., Klein, T., Inoue, M., Hojo, Y., Bessho, Y., Tano, J., Lee, R., Kageyama, A., Kretz, S. H., Hermening , S., Isenmann , I., Liljekvist-Larsson, K., Johansson, B., Reidel, W., Orisme, T., Goldmann, W. C., Smith, U., Wolfrum, H., Xin, J. A., Yannazzo, R. S., Duncan, E. V., Gregg, M., Singh, P., Koulen, S. A., Johnston, D. C., Tang, D. C., Lo, A. K., McAllister, K. a. t. z., LC, L. E., Rajagopalan, J. S., Malter, A. K., McAllister, W. -. B., Gan, J., Grutzendler, W., Wong, R., Wong, J., Lichtman, H., Sato, S., Hattori, S., Kawamoto, I., Kudoh, A., Hayashi, I., Yamamoto, G., Zhang, M. E., Selzer, R. L., Sohn, M. T., Murray, K., Schwarz, J., Nyitray, P., Purray, A. P., Franko, J. A., O'Brien, M., Holt, G., Whiteside, S. C., Lummis, M. H., Hastings, L., Klimaschewski, W., Nindl, M., Pimpl, P., Waltinger, K., Pfaller, P., Kettunen, J., Demas, C., Lohmann, N., Kasthuri, Y., Gong, R. O., Wong, N., Gamper, M. S., Shapiro, M. J., Wirth, P., Wahle, J., O'Brien, S. C., Lummis, A. M., Benediktsson, S. J., Schachtele, S. H., Green, M. E., Dailey, L., Chow, E. M., Levine, T. A., Reh, R. L., Rockhill, F. J., Daly, M. A., MacNeil, S. P., Brown, R. H., Masland, W., Sun, N., Li, S., He, W., Sun, N., Li, S., He, R. C., Stacy, R. O., Wong, E., Strettoi, V., Pignatelli, C., Rossi, V., Porciatti, B., Falsini, V., Pignatelli, E., Strettoi, V. P., Connaughton, D., Graham, R., Nelson, P. H., Edqvist, F., Hallbook, T. C., Jakobs, R. T., Libby, Y., Ben, S. W., John, R. H., Masland, Y., Qin, G., Xu, W., Wang, R., Roizenblatt, J. D., Weiland, S., Carcieri, G., Qiu, M., Behrend, M. S., Humayun, A., Koizumi, G., Zeck, Y., Ben, R. H., Masland, T. C., Jakobs, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explant+culture+of+adult+goldfish+retina:+a+model+for+the+study+of+CNS+regeneration.">Explant culture of adult goldfish retina: a model for the study of CNS regeneration.</a> Brain Res. 161, 39-55 (1979).</li></ol>

1. Что я могу сделать с помощью этого метода? <ul><li> Смотрите морфологии клеток ясно. </li><li> Этикетка клетки для записи на них. </li><li> Гиперэкспрессией белка, как и PSD95, но и других белков, которые модулируют ответ клетки, например, ионные каналы, рецепторы. </li><li> Экспресс тормозных РНК. </li></ul> 2. Как долг...

organotypic culture of adult rabbit retina

Органотипической системы культуры функциональных нервных тканей являются важными инструментами в нейробиологических исследований. В идеале такая система должна быть совместима с методов визуализации, генетические манипуляции, и электрофизиологические записи. Здесь мы приведем простой межфазной культуры тканей система для взрослых сетчатки кролика, что не требует никакого специального оборудования и минимального обслуживания. Мы показываем, рассечение и инкубации сетчатки кролика и частица-опосредованного переноса генов плазмид кодирования GFP или различных субклеточных маркеров в ганглиозных клеток сетчатки. Кролик сетчатки культурный таким образом, может быть поддержана в течение 6 дней с очень небольшим изменением общей структуре анатомических или морфологии отдельных ганглиев и amacrine клеток.

Watch this Scientific Journal Video about Organotypic Culture of Adult Rabbit Retina at JoVE.com

Organotypic Culture of Adult Rabbit Retina

В этой статье показано вскрытие и инкубации сетчатки кролика и частица-опосредованного переноса генов плазмид кодирования GFP или различных субклеточных маркеров в ганглиозных клеток сетчатки.

Органотипической культуры взрослых сетчатки кролика

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

organotypic-culture-of-adult-rabbit-retina

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Biology

11.9K Views.  MGH - Massachusetts General Hospital. В этой статье показано вскрытие и инкубации сетчатки кролика и частица-опосредованного переноса генов плазмид кодирования GFP или различных субклеточных маркеров в ганглиозных клеток сетчатки.

Video: Organotypic Culture of Adult Rabbit Retina

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

Горизонтальное Подготовка Кусочек сетчатки

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Органотипической культуре Кусочек E18 Мозги крысы

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Первичные культуры взрослых Миоциты сердца крысы

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

В естественных условиях подобные органотипической мышей сетчатки культуры Wholemount

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Взрослые и эмбриональные скелетных мышц Microexplant культуры и выделение скелетных мышц стволовых клеток

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Выделение и культуры взрослых Эпителиальные стволовые клетки из человеческой кожи

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

В естественных условиях электропорации Morpholinos в Retina взрослых данио рерио

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Выделение и культура Индивидуальные мышечных волокон и их сателлитные клетки от взрослых скелетных мышц

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

Изоляция, культура и функциональная характеристика взрослой мыши Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Выделение и Культура Стоматологическая эпителиальных стволовых клеток из взрослых мышей резца

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...