ES株化細胞について
「Pluripotency Factors in ES Cells Regulate Differentiation into Germ Layers」
という論文のSupplemental Informationに当たるのですが、以下の文はOct4-mCitrineトランスジェニックES株化細胞の作製方法が書かれた分になります。
An insert containing homology to 50bp upstream of the Oct4 stop codon, the glycine/serine-rich linker, mCitrine cDNA, the native 30 UTR and pA site of Oct4, the loxP-PGK-gb2-neo-loxP selection cassette (Gene Bridges), and homology to 50bp downstream of the Oct4 30 UTR was generated by fusion PCR and validated by sequencing the final amplicon.
This fusion insert was electroporated into BAC strain RP24-248K18 (CHORI) harboring Oct4, and Red/ET Recombination was performed.
Properly modified BAC clones were screened by PCR.
A BAC fragment containing the distal and proximal enhancers of Oct4 (Minucci et al., 1996; Tesar et al., 2007), the Oct4 ORF, the fusion insert, and 2.1kb of 30 homology was subcloned into pColE1 using Red/ET Recombination (Figure S4B).
DNA linearized by I-PpoI was electroporated into V6.5 ESCs, and colonies were selected on G418 (200 mg/mL).
Clones that stably retained mCitrine fluorescence in pluripotency maintaining conditions for many passages were selected and further characterized by immunofluorescence, PCR, Southern blotting, and flow cytometry.
この文を訳してみたのですが、この実験の原理が全く分からないので理解することができませんでした。なぜBACを使うのか？なぜloxP-PGK-gb2-neo-loxPカセットが必要なのか？など基本的なことが分かりません。
分かる方教えてください。よろしくお願いします。
ちなみに添付画像はFigure S4Bです。
