In Utero（子宮内）胎児へのエレクトロポレーションによる遺伝子導入

NEPA21エレクトロポレーション装置

Sピンセット電極

ｹｰﾌﾞﾙ･ﾌｯﾄｽｲｯﾁ

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文献

アプリケーション

エレクトロポレーション法によるマウス子宮内胎仔脳への遺伝子導入

１）準備する機械・器具

２）In Uteroエレクトロポレーション

ICRマウスを用いる場合の電気設定条件

妊娠日数	電極径	電圧	パルスオン	パルスオフ	回数
12.5日	3 mm	33 V	30 msec	970 msec	4
13.5日	5 mm	30 V	50 msec	950 msec	4
14.5日	5 mm	33 V	50 msec	950 msec	4
15日～	5 mm	35 V	50 msec	950 msec	4

導入効率よりダメージを少なくする方を優先する場合は、回数を2回に設定。導入効率よりダメージを少なくする方を優先する場合は、回数を2回に設定。

この時、実測の電流値は30mA～60mAとなる（実測値は、電気パルスの後、機械のディスプレーに表示される）。ただし、電極の当て方や子宮の濡れ具合によってもこの値は容易に変動する。まずは、上記の条件で、電流値がこの範囲に入るように電極間の距離や、陰極の子宮に接する面積を検討する。調整しきれない場合には、電圧の設定を変える。

３）GFP発現ベクターを導入した例

胎生14.5日目のICRマウスにおいて、両半球の側脳室にCAG-EGFPを注入し、33V、50msecの電気パルスを4回与えた。 3日後（胎生17.5日目）に胎仔をかん流固定して脳を摘出し、蛍光実体顕微鏡で観察した (図A) 。これらの脳において、一方の大脳半球では内側が、もう一方の半球では外側が遺伝子導入され、GFPによる蛍光をはっきりと認めることができた。
また、これらの脳から凍結切片をつくり、GFPの蛍光を共焦点レーザー顕微鏡で観察した（図B）。脳室帯で遺伝子導入されたGFP陽性細胞が脳の表層側へと移動し、中間帯や皮質板にまで進入してきていることが観察された。矢頭は脳室帯と中間帯、もしくは中間帯と皮質帯との境界を示す。破線は組織の境目を示す。 VZ：脳室帯, IZ：中間帯, CP：皮質板

慶応義塾大学医学部解剖学教室田畑秀典先生・仲嶋一範先生提供
※羊土社「必ず上手くいく遺伝子導入と発現解析プロトコール」転載

In Utero（子宮内）胎児へのエレクトロポレーションによる遺伝子導入

アプリケーション

エレクトロポレーション法によるマウス子宮内胎仔脳への遺伝子導入

１）準備する機械・器具

２）In Uteroエレクトロポレーション

３）GFP発現ベクターを導入した例

文献

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