pCAT^-Basic

The pCAT®-Basic vector (Promega) contains the the chloramphenicol acetyl transferase gene but lacks eukaryotic promoter and enhancer sequences. Expression of CAT activity in cells transfected with this construct is dependent on insertion o f a functional promoter into the multiple cloning site upstream of the CAT gene. Enhancer elements can also be cloned into the BamHi site downstream of the CAT gene (Figure 2.1C).

Figure 2.1: Commercially available plasmids Hinc ( 1 ) ATG Kpn\ lacZ 7 1 2 8 bp AmpR S V 4 0 o r i / Early p r o m o te r 4 0 codons g p t codons trp S BamH\ Am pR S V 4 0 4 3 4 4 bp C A T HindWl A ) p C H l 1 0 C ) p C A T - B a s i c S V 4 0 p AmpR C A T 4 7 5 2 bp S V 4 0 e n h a n c e r B ) p C A T - C o n t r o l KEY gpt= E.coli gpt promoter

trp S= structural E.coli tryptophanyl- tRNA synthetase gene

AmpR= ampicillin resistance gene lac Z= the gene for beta galactosidase SV40p= SV40 promoter region

CAT= gene for chloramphenicol acetyltransferase

2.10.4 ppPASe9

ppPASe9 is a muscle-specific expression construct that was previously generated in the laboratory by Ricarda Steinbrecher. It contains the promoter element of the rabbit p~ cardiac myosin heavy chain gene, originally described by Cribbs et a l in 1989 (Dr.Patrick Umeda, University of Alabama, Birmingham, USA), a 920 bp enhancer element of the rat myosin light chain 1/3 gene locus originally described by Donoghue et a l 1988 (Dr.Nadia Rosenthal, Boston, MA, USA), and a 45 bp 3' polyadenylation signal sequence, designed according to consensus sequence requirements (McLauchlan, 1985), cloned into the HinâMl, BamRl and between the SalV and Bam Rl sites of pUC19 respectively. The resulting construct ppPASe9 is shown in Figure 2.2 A and has been described previously (Steinbrecher et al, 1993).

2.10.5 pCDMS/hfVII

The cDNA for hfVII, was supplied by Dr. John McVey (Haemostasis Research Group, Royal Postgraduate Medical School, UK). It was cloned into the EcoBl site of expression vector pCDM8 (Invitrogen BV, De Schelp 12, 9351 NV Leek, The

Netherlands) which had had its 358bp stuffer replaced with a linker which inserts EcoBJ and BamHl restriction sites (Figure 2.2B). The human factor VII insert is flanked by Xhol restriction enzyme sites in the pCM8 vector and therefore can be removed by

digesting with Xhol.

Figure 2.2: Plasm ids used for the production o f a human factor V II expression construct MHCp H / n d l l l /S a /I ,PA S RBS BamHl 4 5 4 0 bp AmpR M LC920 BamHl B ) p C D M B /h F V II P CMV supF hfVII M l 3 ori 6 8 6 0 bp ColEI - EcoRI Xho\ SV40 nri ^ S V 4 0 Intron/pA KEY

AmpR= ampicillin resistance gene MHCp= myosin heavy chain promoter MLC920= myosin light chain enhancer RBS= ribosomal binding site

PAS/pA= polyadenylation signal sequence hfVII= human factor VII cDNA

pCMV= human cytomegalovirus promoter M13 ori= m l3 origin of replication

ColEl= origin of replication for growth in E.coli SupF= suppressor tRNA for maintenance in E.coli

SV40/ polyoma ori= origin of replication for episomal replication in cells expressing the SV40 large T antigen or latently infected with the polyoma virus SV40 intron /pA= transcription termination and RNA processing signals from SV40

2.10.6pcDNA3.1/His C & pcDNA 3.1/HisB/lacZ

pcDNA3.1/His C is a fusion vector (Invitrogen®) and contains the human cytomegalovirus immediate-early (CMV) promoter, an N-terminal polyhistidine tag to permit purification of recombinant protein on metal-chelating resin and an Anti- Xpress™ epitope tag which allows detection of the recombinant protein with the Anti- Xpress™ antibody (Figure 2.3A, Invitrogen®). The N-terminal polyhistidine tag can be removed from the recombinant protein using an enterokinase which has a cleavage site in the vector immediately after the polyhistidine residues. The vectors pcDNA3.1(-)/Myc-His and pcDNA 3.1/His (Invitrogen®) are supplied with three different versions (A, B & C), of the multiple cloning site to allow cloning o f inserts in the three different reading frames. pcDNA 3.1/HisB//acZ is the vector version B with a 3.2 kh fragment containing the p-galactosidase gene cloned in frame with the N- terminal peptide. It is used as a positive control for transfection, expression and purification.

2.10.7pcDNA 3.1/Myc-His & pcDNA3.1(-)Myc-HisAacZ

pcDNA3.1(-)/Myc-His (Figure 2.3B, Invitrogen®) is a fusion vector containing the human cytomegalovirus immediate-early (CMV) promoter to provide high level expression in a wide range of mammalian cells, a C-terminal polyhistidine tag to permit purification of recombinant protein on Nickel-chelating resin (ProBond'^’^and a myc epitope (c-myc) which allows detection of the recombinant protein with an anti-myc antibody (Invitrogen®, Evan et al, 1985). pcDNA3.1(-)/Myc-His//acZ is the pcDNA3.1(-)/Myc-His vector version A with a 3.2 kb fragment containing the p- galactosidase gene cloned in frame with the C-terminal peptide.

Figure 2.3: Plasmids used for the C and N terminal tagging of mFVII A) pcDNA3.1/HisC

1

^

S V 4 0 ori BGH pA 5 5 0 0 bp \ AmpR S V 4 0 pA B) pcDNA3.1(-)/M yc-His

myc

BGH pA

S V 4 0 ori 5 5 0 0 bp

S V 4 0 p

KEY

^CMV/CMVp~ cytomegalovirus immediate-early promoter BGH pA= bovine growth hormone polyadenylation signal T”= T7 promoter/priming site

His polyhistidine tag,

term= termination codon from the vector NeoR= neomycin (0418) resistance gene AmpR= ampicillin resistance gene

ColEl= origin of replication for growth in E.coli

EK= enterokinase site for removal of the anti-xpress epitope S\'40 pA= RNA processing signal from SV40

2.10.8 pCIS/mJVII

pCIS/fVII (Dr E Rosen, University of Notre Dame, Indiana USA) has previously been described (Idusogie et al., 1996) briefly the mfVII cDNA cloned into the pBluescript SK' vector with EcoRl was cut out and cloned into pCIS2M (Zhang & Castellino. 1991) with the restriction enzymes Xba\ and Xhol (Figure 2.4).