A restriction site-free cloning method has been developed for inserting a PCR product into a vector flexibly and precisely at any desired location with high efficiency. The method uses a pair of DNA integration primers with two portions. The 3' portion isolates the inserts by PCR, and the 5' portion integrates the PCR products into the homologous region of the vector. For mutagenesis, a third portion of mutation-generating sequences can be placed in between the 3' and 5' portions. This method has been used to clone the E. coli gene that codes for peptidyl-tRNA hydrolase, expressing it as a native protein and as a glutathione S-transferase fusion protein. It was also applied to convert a construct of the E. coli fatty acid biosynthesis protein with an N-terminal hexa-histidine tag into a construct with a C-terminal hexa-histidine tag.
A 3233 base pair (bp) sequence of the 5′-flanking region of the mouse cellular retinoic-acid-binding protein (CRABP) gene is determined. From this region, a 3 kb fragment located 150 bp upstream from the transcriptional initiation site is isolated and fused to a LacZ reporter sequence. Transgenic mouse embryos of this fusion gene show spatially and temporally specific expression of LacZ protein and the expression of this fusion gene at the RNA level is confirmed by RNAase protection assays, which detect specific fusion transcripts in RNA samples from tissues of transgenic mouse embryos. In contrast, transgenic mouse embryos of a shorter fusion gene containing only 583 bp from the same upstream region of the mouse CRABP gene fused to the same reporter sequence show no LacZ activities. Thus, it is concluded that the 3 kb sequence, but not the 583 bp sequence, of the mouse CRABP gene contains information for its temporally and spatially specific expression in mouse embryos.
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