3.2 METHODS 
3.2.1. Construction of vector producing cell lines 
3.2. 1.1. CONSTRUCTION OF HUMAN y- INTERFERON RETROVIRAL PROVECTOR PLASMID, 
PHUy-IFN. A cDNA for human y-IFN was obtained by PCR. RNA was isolated from PHA- 
stimulated Jurkat T cells by guanidinium thiocyanate extraction followed by ultracentrifugation 
through a CsCl cushion (45). RNA was reverse- transcribed in vitro as described (46) and a gene- 
specific oligonucleotide pair was used to amplify y-IFN cDNA by the polymerase chain reaction 
using Tac polymerase (47). The PCR DNA was repaired with T4 DNA polymerase and Klenow, 
kinased with T4 Polynucleotide Kinase, and cloned into the HinC II site of SK + plasmid 
(Stratagene) treated with calf intestinal phosphatase. In the sense orientation, the 5' end of the 
cDNA was adjacent to the Xho 1 site of the SK+ polylinker and the 3' end adjacent to the Cla 1 
site. Sequencing of phy-IFN, the retroviral vector, revealed the presence of a one base pair 
deletion within the phy-IFN gene. This deletion was reversed using a multi-step PCR procedure. 
The y-IFN gene in the appropriate orientation was removed from the SK + plasmid by digesting 
DNA with Xho I and Cla I and cloned into the MoMLV-based retroviral vector, KT-3 (manuscript 
in preparation). Briefly, KT-3 contains the LTR and packaging sequences from vector N2 (48), 
except that the ATG translational initiation codon of MLV gag (present in the packaging sequence, 
T) was mutated to ATT in order to prevent undesired translation of these sequences. The plasmid 
contains unique Xho I and Cla I cloning sites immediately after the packaging signal and before the 
Neo r gene (expressed from the SV40 early promoter). KT 3 plasmid was digested with Xho I and 
Cla I and ligated with the Xho I-Cla I y-IFN fragment. Positive clones were identified by 
restriction mapping. Subsequent sequencing of the y-IFN cDNA insert in the provector plasmid 
confirmed the exact identity of the cDNA. The overall structure of Huy-IFN provector is described 
in Fig 2. The sequence and structure of pHuy-IFN is presented in Appendix H. 
3.2. 1.2. Construction of Murine y-IFN retroviral pro vector, pMuy-lFN. A mouse y- 
IFN cDNA cloned into the EcoRI site of pUC18 (49) was obtained. The y-IFN cDNA was 
retrieved by EcoRI digestion and the isolated fragment was cloned into the EcoRI site of 
phosphatase-treated pSP73 vector. The orientation of the cDNA was verified by using appropriate 
restriction enzyme digestion and DNA sequencing. In the sense orientation, the 5' end of the 
cDNA was adjacent to the Xho 1 site of the pSP73 polylinker and the 3' end adjacent to the Cla 1 
site. Xho 1 -Cla 1 fragments containing the murine y-IFN cDNA were retrieved from pSP73 and 
cloned into the Xhol-Cla 1 site of KT-3 (see 3.2.1. 1 above). See Fig 2 for a diagrammatic 
representation of the Muy-IFN pro vector. 
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