C. Vectors and Viruses 
• 1. Vectors for transfer and expression of human LDL receptor 
The proviral components of these vectors are mainly derived from the previously 
described vector DOI (104) are presented as follows: The parent vector BA-(h)LDLR has been 
described in a previous publication (14). The general structure of the vector will be described 
below. The backbone structure of this vector 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 Xho I site in the 
gag coding region at nucleotide 1560 (with the exception that a Sac II linker was inserted at the 
Hae III site at nucleotide 624; see Ref. 105) The plasmid also contains wild-type Mo-MLV 
sequences from the Cla I site at nucleotide 7674 (which was converted to Bam HI site with 
synthetic linkers) to the end of the 3' LTR. Sequences containing the viral enhancer elements of 
the 3' LTR from the Pvu II site at nucleotide 7933 to the Xba I site at nucleotide 8111 have been 
deleted. In addition to these sequences, there are flanking mouse genomic DNA and pBR322 
sequences (spanning the Hind III site to the Eco Rl site). The initial promoter 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 (106). The Mbo I site was converted to a Bam HI site and the modified 
p-actin fragment was cloned into the parent vector. The LDL receptor coding sequences were 
derived from a 2.6 kb Hind III fragment of a full-length cDNA fragment (Ref. 54; provided by D. 
Russell, J. Goldstein, and M. Brown). The Hind III sites were converted to Bel I sites and the 
cDNA was cloned into the Bam HI site of the vector. This cDNA fragment contains the entire 
coding sequence with 13 base pairs (bp) of 5' untranslated sequence (AGCTTAATACACA) and 5 bp 
of 3' untranslated (ATCAG). An enhancer was introduced into the Xho I site of BA-(h)LDLR 
vector. These sequences were derived from an area 5' to the immediate early (IE) gene of 
human cytomegalovirus [from Spe I (at -580 of IE gene) to Pst I (site in vector sequence) of 
CDM8, Ref. 107] were subcloned into PUC19. A portion containing IE enhancer sequences was 
removed on a Xho I (from polylinker) to Nco I (-220 of the IE gene) fragment (for numbering 
of IE enhancer see Ref. 108). Synthetic linkers were used to convert the Nco I site to a Xho I 
site and the modified fragment was cloned into the unique Xho I site of BA-(h)LDLR located 5' to 
the p-actin promoter. This new vector is called CMV-BA-(h)LDLR. The structure of this 
vector is presented in Fig.2. 
2. Description of the CMV-BA (h) LDLR retroviral sequences 
The complete nucleotide sequence of the proviral component of this plasmid is currently 
being determined by Lark Sequencing Technologies Inc. Sequence determination is being 
performed in compliance with FDA/EPA Good Laboratory Practices. The region to be sequenced 
has been subcloned as two overlapping restriction fragments into pBluescript II (Stratagene) 
and pGem5Zf (Promega). Nested deletion clones are being generated in both directions for each 
of the subclones using a modified exo III/S1 nuclease procedure. These deletion clones will be 
size selected to provide complete coverage of each strand and sequenced using the 
dideoxynucleotide termination procedure. Internal sequencing primers will be synthesized and 
used to close gaps between contigs and to fill in any single-stranded regions. Anticipated 
completion of this project is November 1,1991. 
3. Isolation of the LDL receptor virus producing cell line 
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Viral-producing cell lines were isolated for each vector using the amphotropic 
packaging cell line v P-Crip (90). 'F-Crip is an amphotropic packaging cell line constructed by 
Dr. Olivier Danos in Dr. Richard Mulligan's laboratory. A brief review of the construction of 
this cell line is provided below. 'F-Crip cell line was constructed by transfecting the gag-pol 
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Recombinant DNA Research, Volume 15 
