A 1.2 kb StuI-SnaBI pH5-gfpzeo expression cassette from pG06dH5gz was blunt cloned in both leftward and rightward orientations into the Klenow-filled XbaI site in pMSΔ102 to yield plasmids pMSΔ102H5GZ.1, -.4, respectively.
Thus, this region was deleted from piggyBac vectors containing the constitutive GalT gene in the leftward or rightward orientations (Fig. 7, vectors B and D) and GalT expression levels induced by these new derivatives were compared to those induced by controls containing the intact promoter.
This finding means that an individual participant was more likely to call a foul when seeing a picture in its leftward compared to rightward orientation, even though the two stimuli were otherwise identical.
The activity levels induced by the piggyBac vectors containing the heterologous genes in the rightward orientation were significantly (5X to over 35X) higher.
For the observations on posture the 3D coordinates were defined as follows: the positive x direction was the forward orientation, the positive y direction was the rightward orientation, and the positive z direction was the upward orientation.
Similarly, vectors encoding GalT, ST6GalI, and ST3GalIII genes in the rightward orientation induced about 8X, 8X, and 3X more activity, respectively, than vectors encoding these genes in the leftward orientation (Fig. 5, columns A-B).
Thus, together with our previous results, these results suggested that the position effect observed with our new piggyBac vectors results from the activated expression of heterologous genes cloned in the rightward orientation, irrespective of the transcriptional control element.
Probe R containing the DNA sequence at nt 576 67 (complementary to RNAs transcribed in the rightward orientation) hybridized to the 350-nt RNA (ORF3) and the internal control band.
This effect was not restricted to mammalian glycosyltransferase genes, as piggyBac vectors containing SAS and CMP-SAS genes in the rightward orientation induced about 3X and 4X higher sialic acid and CMP-sialic acid contents, respectively, than those containing these same genes in the leftward orientation (Fig. 5, Column C).
For example, cells transfected with the piggyBac vector containing the GnTII gene in the rightward orientation (vector A in Column A, Fig. 5) had about 4.5X more GnTII activity than cells transfected with the piggyBac vector containing this same gene in the leftward orientation (vector B in Column A, Fig. 5).
Constitutive and inducible piggyBac vectors containing the GalT gene in the leftward orientation with no downstream copy of the 3'-TRDID sequence (Fig. 10, vectors B and E) were used as negative controls and a constitutive piggyBac vector containing the GalT gene in the rightward orientation (Fig. 10, vector A) was used as a positive control.
