For optimum expression of the inserted gene, there are several important points to consider when cloning:
First, the level of expression of the inserted gene is not only influenced by proviral sequences within the LTR, but also by sequences within the pol region. The more recently developed RCAS vectors containing the pol region from the Bryan high-titer strain of RSV, known as RCASBP (BP stands for Bryan pol), express introduced genes at higher levels (17) than do the original RCAS vectors and may be preferable for cloning (see also Note 1).
Second, the gene of interest should be cloned into the unique ClaI restriction site of the RCASBP retroviral vector via an intermediate cloning step using the adapter plasmids pCla12Nco (1) or pSLAX12 (16). pSLAX12 was made by transferring the ClaI to ClaI fragment from pCla12Nco into a pBluescript (Stratagene) vector and has the increased advantage of having a higher copy number than does pCla12Nco. In addition, commercially available T3 and T7 primers can readily be used for sequencing pSLAX12 subclones. These adapter plasmids contain several restriction sites flanked by two ClaI sites, and not only facilitate cloning, but also contain part of the 5'-untranslated region of the src oncogene, the inclusion of which has been found to enhance considerably the level of expression of the inserted gene.
Third, the coding sequences of the gene of interest should be inserted in frame with the ATG of the NcoI site within the adapter polylinker to avoid altering the N-terminus of the encoded protein. Inclusion of untranslated 5'- and 3'-regions of the gene of interest should be avoided, since they can adversely affect subsequent transcriptional and/or translational efficiency.
1. Appropriate insert fragments to be subcloned can be generated by either restriction digests or by PCR, and subcloned into the adapter plasmid by standard cloning procedures. When using pCla12Nco, recombinants must be identified by radioactive screening and/or PCR as LacZ blue-white selection is not possible. If the insert fragment is generated by PCR, restriction sites can be added to the oligos to facilitate cloning. In the latter strategy, an NcoI site can be conveniently introduced (if not already present) at the initiator ATG site, thus allowing the fragment to be directly cloned in frame into the NcoI site of the adapter polylinker. Providing that the second codon begins with a guanine, such mutagenesis will not alter the encoded sequence. If the second codon does not begin with a guanine, a more complicated cloning strategy, such as that outlined by Morgan and Fekete (16) is required. For efficient cutting of the PCR product, at least two bases (preferably four for NcoI) should be included in the oligo 5' to the restriction sites. To avoid errors in the PCR product, the oligos should be very pure and a thermostable polymerase, which has proof-reading ability (e.g., PFU polymerase, Promega), should be used. Finally, once subcloned, the sequence of the PCR product and/or inserted fragment should be thoroughly checked to ensure that no mutations have been introduced (see Note 2).
2. The ClaI to ClaI fragment from the adaptor plasmid containing the inserted coding sequences is then isolated by partial or complete restriction enzyme digestion and subcloned into the appropriate RCASBP and/or RCANBP vectors by standard cloning procedures. Again recombinants must be identified by radioactive screening and/or PCR analysis. Orientation of the insert may be determined by restriction digest analysis. Alternatively, the inserts may be sequenced using appropriate oligonucleotide primers (16) (see Note 3).
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