pUC18 and subsequently recovered as a BamHI/Hindlll fragment which was treated with T4 DNA 
polymerase prior to blunt end ligation into the vector pNAss. The E.Coli p-galactosidase gene 
from pC4AUG ( McGregor et al. f 1987) was excised as a 3530bp EcoRI - Xbal fragment and, 
following the addition of Notl linkers, cloned into the unique Notl site of the vector. 
The entire minigene containing the CMV promoter, lacZ gene, and SV40 polyA were excised from 
pCMV-p on an Sphl fragment. This fragment was treated with Klenow and ligated with Bell 
linkers. The pAdBglll vector was digested with Bglll and treated with calf intestinal phosphatase 
before the Bell linkered minigene was cloned in direct orientation into the Bglll site of pAdBglll. 
See Figure 17, panel C for overview. 
Step 3. Construction of pAd.CMV-lacZ. The purpose of this construction was to remove the CMV 
promoter and lacZ gene from the pAdCMV-lacZ vector and introduce a fragment containing a 
portion of the CMV enhancer, the chicken p-actin promoter, the full-length CFTR minigene and 
a small portion of retroviral sequences from Mo-MLV. A brief description of the retroviral 
vector from which the CFTR gene was described is provided below. 
The backbone structure of this vector called pBA-CFTR includes an intact 5'LTR of Moloney 
murine leukemia virus (Mo-MLV) with additional Mo-MLV sequences between the 5' LTR and 
the internal promoter spanning nucleotide 146 at the border of U5 to the natural Xhol site in 
the gag coding region at nucleotide 624; see Van Beveren er all, 1985). The plasmid also 
contains wild-type Mo-MLV sequences from the Clal site at nucleotide 7674 (which was 
converted to a BamHI site with synthetic linkers) to the end of the 3' LTR. Sequences containing 
the viral enhancer elements of the 3' LTR from the Pvull site at nucleotide 7933 to the Xbal site 
at nucleotide 81 1 1 have been deleted. In addition to these sequences, there are flanking mouse 
genomic DNA and pBR322 sequences (spanning the Hindlll site to the EcoRI site). The initial 
promote used in this vector was derived from a Xho I to Mbo I fragment of the chicken p-actin 
gene spanning nucleotides -266 to +1 (Kost et al., 1 983). The Mbol site was converted to a 
BamHI site and the modified p-actin fragment was cloned into the parent vector, called pgagBA. 
The human CFTR coding sequences were derived from a 4.6 kb Sacl fragment which contains the 
complete coding sequence and small 5' and 3' untranslated regions (Riordan et al., 1989). The 
Sacl sites were converted to Bell sites and the modified fragment was cloned into the BamHI site 
of pgagBA. This vector is called pgagBA-CFTR. An enhancer was introduced into the Xhol site of 
this vector . Thess sequences were derived from an area 5' to the immediate early (IE) gene of 
human cytomegalovirus [from Spel (at -580 of IE gene) to Pstl (site in vector sequence) of 
CDM1, Seed et al., 1987] were subcloned into PUC19. A portion containing IE enhancer 
sequences was removed on a Xhol (from polylinker) to Ncol (-220 of the IE gene) fragment 
(for numbering of IE enhancer see Boshart et al., 1985). Synthetic linkers were used to 
convert the Ncol site to a Xho I site and the modified fragment was cloned into the unique Xhol 
site of pgagBACFTR located 5' to the p-actin promoter. This new vector is called pCMV-BA- 
CFTR. 
The retroviral vector pCMV-BA-CFTR was digested with Xho I and Nhe I to release a fragment 
(BA-CFTR) containing the chick p-actin promoter, the 4.6 kb human CFTR cDNA and a small 
portion of retrovirus-specific sequences. The plasmid pAd.CMV-lacZ was cut with Sna Bl and 
Not I to remove CMV promoter and lacZ structural gene, and the plasmid backbone retaining the 
CMV enhancer and the SV40 polyadenlyation signal was blunted and ligated with the blunted BA- 
CFTR fragment to form the plasmid pAd.CB-CFTR. For a schematic representation of this please 
see Figure 17, panel D. 
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Recombinant DNA Research, Volume 16 
