The enhancer and protein trap cassettes are in opposite orientation, with an intervening mini Tol2 element, which can act as a launching pad for Tol2 transposase-mediated remobilizations in the vicinity of the targeted DsDELGT4 insertion sites, for further insertional mutagenesis.
Insertions "available" for further use or analysis fall under three categories: "alive" (line established with mice available in small numbers), "cryopreserved" (either as embryos or sperm) and "new" (usually corresponding to a new insertion, with only the founder animal).
The synthesized gene was flanked by compatible but non-regenerative restriction sites (BamHI and BglII) to enable the insertion of further synthetic genes for extension of the spidroin content.
The seeds from these plants, i.e. T0 seeds, were germinated on kanamycin-containing medium and on the basis of segregation analysis and genomic Southern blot; transgenic lines with single transgene insertion were selected for further analyses.
Among the kanamycin-resistant independent transformants, only plants containing a single insertion were selected for further characterization.
Fifty-nine independentransformantsts were recovered and three high-expressing lines containing single insertions were selected for further analysis (35S ABF3-48, -57, and -59).
The insertion of the restriction endonuclease sites provides a convenient method for further insertions of purification and/or epitope tags into permissive domains.
In the T1 generation two single-copy insertion lines were selected for further expression analysis (lines 12 and 41).
Transformed lines were selected on kanamycin and single-insertion lines were selected for further analyses.
Importantly, the inward facing BsmBI site on the last introduced oligonucleotide remains intact upon ligation and is available for further insertions.
