Name: a cre-lox p recombination approach for the detection of cell fusion in vivo
Uploaded: 2012-01-04T12:00:00
Description: Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

Авторы хотели бы поблагодарить д-ра Пейман Хеммати (Департамент медицины, Университет Висконсин-Мэдисон) для предоставления щедро H1 МСК, и доктор Тим Хакер, доктор Gouqing песни и г-жа Джилл Кох из университета Висконсина сердечно-сосудистой физиологии Основной фонд для выполнения операции мыши. Работа выполнена при поддержке Национального научного фонда через стипендий Исследование Брайана Фримен и НИЗ R21 HL089679.

1. Трансфекция клетки-донора <ol><li> Урожай мезенхимальные стволовые клетки (МСК, полученных из H1 эмбриональных стволовых клеток любезно пожертвовал д-р Пейман Hematti; альтернативно, любого типа клеток какого-либо вида гипотетически предохранитель в естественных условиях могут б...

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Описанный здесь метод позволяет, в первый раз, дискретные идентификации и временной анализ слияния клеток в организме, в том числе мелких животных. Подход сочетает Cre-LoxP рекомбинации с последующей Биофотонный анализа изображений. Подход поддаются отслеживанию не только клеточного сли...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер по каталогу </td><td> Комментарии </td></tr><tr><td> Neon трансфекции системы </td><td> Invitrogen, Карлсбад, Калифорния </td><td> MPK5000 </td><td></td></tr><tr><td> Neon 100 мкл Kit </td><td> Invitrogen, Карлсбад, Калифорния </td><td> MPK10025 </td><td> Содержит R и E буфера </td></tr><tr><td> MEM-порошка </td><td> Invitrogen, Карлсбад, Калифорния </td><td> 12000-022 </td><td></td></tr><tr><td> Эмбриональной бычьей сыворотки (FBS) </td><td> Hyclone, Логан UT </td><td> SH30070.03 </td><td></td></tr><tr><td> B6.C-TG (ЦМВ-CRE) 1Cgn / J </td><td> Лаборатории Джексона, Bar Harbor, ME </td><td> 006054 </td><td></td></tr><tr><td> Трипсин 10X </td><td> Fisher Scientific, Forest Lawn, Нью-Джерси </tг&gt; <td> МТ-25-054-Cl </td><td></td></tr><tr><td> L-глютамин </td><td> Fisher Scientific, Forest Lawn, Нью-Джерси </td><td> 25030-081 </td><td></td></tr><tr><td> D-люциферин </td><td> Caliper Life Sciences, Хопкинтон, М. А. </td><td> 122796 </td><td></td></tr><tr><td> Xenogen Биофотонный-изображений </td><td> Caliper Life Sciences, Хопкинтон, М. А. </td><td> ИВИС спектра </td><td></td></tr><tr><td> Натрий Biocarbonate </td><td> Sigma Aldrich, Сент-Луис, Миссури </td><td> S6014-500G </td><td></td></tr><tr><td> Номера для незаменимых аминокислот </td><td> Invitrogen, Карлсбад, Калифорния </td><td> 11140-050 </td><td></td></tr></tbody></table>

a cre-lox p recombination approach for the detection of cell fusion in vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Метод для отслеживания слияния клеток в живом организме с течением времени описывается. Подход использует Cre-<em&gt; LoxP</em&gt; Рекомбинации, чтобы побудить люциферазы выражение на слияние клеток. Люминесцентный сигнал, генерируемый могут быть обнаружены в живых организмов, использующих Биофотонный систем визуализации с чувствительностью обнаружения ~ 1000 клеток в периферических тканях.

Cre-LOX P Рекомбинация подход для обнаружения сотовых Fusion В Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

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A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

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