Preparation of competent bacterial cells

A single bacterial colony of Escherichia coli DH5α was inoculated from an LB agar plate into 10 ml LB and incubated overnight at 37°C with shaking. The culture was

Figure 2.1 Schematic representation of the pdlNotI’MCS’F lentivirus vector. Vector diagram of pdlNotI’MCS’F lentivirus vector showing approximate positions of the SFFV promoter, multiple cloning site, IRES and puromycin resistance genes (pac). Important restriction sites are shown. LTR – long terminal repeat; psi – Psi- sequence; RRE – Rev-responsive element; cPPT – HIV central polypurine tract; SFFV – Spleen focus forming virus promoter; MCS’F – Multiple cloning site; IRES – Internal ribossomal entry site; WPRE – woodchuck hepatitis virus post-

Figure 2.2 Schematic representation of the pHR-SIN-CSGWdlNotI lentivirus vector. Vector diagram of pHR-SIN-CSGWdlNotI lentivirus vector showing approximate positions of the SFFV promoter and eGFP gene. Important restriction sites are shown. LTR – long terminal repeat; psi – Psi-sequence; RRE – Rev-responsive element; cPPT – HIV central polypurine tract; SFFV – Spleen focus forming virus promoter; eGFP – enhanced green fluorescent protein; WPRE – woodchuck hepatitis virus post-transcriptional regulatory element.

Figure 2.3 Schematic representation of the pGL3-Mx1P expression vector. Vector diagram of pGL3-Mx1P vector showing approximate positions of the Mx1 promoter, luciferase and ampicillin resistance genes. Important restriction sites that were used in cloning strategies are shown. MCS – Multiple cloning site, PMx1 – Mx1 promoter; luc – luciferase gene; amp – ampicillin resistance gene.

Figure 2.4 Schematic representation of the pGEM-T Easy expression vector (Promega Ltd, United Kingdom).

Vector diagram of pGEM-T Easy expression vector showing positions of the multiple cloning site and the ampicillin resistance gene. Important restriction sites are shown.

Figure 2.5 Schematic representation of the pdl Mx GIPSE lentivirus vector. Important restriction sites are shown.

The pdl Mx GIPSE vector contains both the eGFP and the puromycin resistance (pac) genes under the control of the IFN-inducible murine Mx1 promoter. The IRES element allows for the expression of both eGFP and pac in response to the Mx1 promoter. Mx GIPSE stands for Mx1 promoter, eGFP, IRES, puromycin resistance and IFN-selectable. LTR – long terminal repeat; psi – Psi-sequence; RRE – Rev- responsive element; cPPT – HIV central polypurine tract; PMx1 – Mx1 promoter; eGFP – enhanced green fluorescent protein; IRES – Internal ribossomal entry site; WPRE – woodchuck hepatitis virus post-transcriptional regulatory element.

Figure 2.6 Schematic representation of the pdl Mx TIPSE lentivirus vector. The pdl Mx TIPSE vector contains both the HSV-tk and the puromycin resistance (pac) genes under the control of the IFN-inducible murine Mx1 promoter. The IRES element allows for the expression of both HSV-tk and pac in response to the Mx1 promoter. Mx TIPSE stands for Mx1 promoter, tk, IRES, puromycin resistance and IFN-selectable. LTR – long terminal repeat; psi – Psi-sequence; RRE – Rev-

responsive element; cPPT – HIV central polypurine tract; PMx1 – Mx1 promoter; tk –thymidine kinase gene; IRES – Internal ribossomal entry site; WPRE – woodchuck hepatitis virus post-transcriptional regulatory element.

Figure 2.7 Schematic representation of the pdl’PIV5V’bla lentivirus vector.

Vector diagram of pdl’PIV5V’bla lentivirus vector showing approximate positions of the SFFV promoter, multiple cloning site, PIV5 V, IRES and blasticidin genes. Important restriction sites are shown. LTR – long terminal repeat; psi – Psi- sequence; RRE – Rev-responsive element; cPPT – HIV central polypurine tract; SFFV – Spleen focus forming virus promoter; MCS’F – Multiple cloning site; PIV5V – V gene of PIV5; IRES – Internal ribossomal entry site; blasticidin – blasticidin resistance gene; WPRE – woodchuck hepatitis virus post-transcriptional regulatory element.

Figure 2.8 Schematic representation of the pdl’HA-E3L’pac lentivirus vector. Vector diagram of pdl’HA-E3L’pac lentivirus vector showing approximate positions of the SFFV promoter, HA-tagged E3L, IRES and pac genes. Important restriction sites are shown. LTR – long terminal repeat; psi – Psi-sequence; RRE – Rev- responsive element; cPPT – HIV central polypurine tract; SFFV – Spleen focus forming virus promoter; HA-E3L – HA-tagged E3L gene of VV; IRES – Internal ribossomal entry site; pac – puromycin resistance gene; WPRE – woodchuck hepatitis virus post-transcriptional regulatory element.

Figure 2.9 Schematic representation of the pdl’Flag-E3L’pac lentivirus vector. Vector diagram of pdl’Flag-E3L’pac lentivirus vector showing approximate positions of the SFFV promoter, Flag-tagged E3L, IRES and pac genes. Important restriction sites are shown. LTR – long terminal repeat; psi – Psi-sequence; RRE – Rev-responsive element; cPPT – HIV central polypurine tract; SFFV – Spleen focus forming virus promoter; Flag-E3L – Flag-tagged E3L gene of VV; IRES – Internal ribossomal entry site; pac – puromycin resistance gene; WPRE – woodchuck hepatitis virus post-transcriptional regulatory element.

54 diluted 100 times in LB and grown until the OD600nm reached 0.400 to 0.600 units. 50 ml

cultures were then incubated on ice for 30 min and pelleted by centrifugation at 2800 rpm for 5 min at 4°C. Cells were ressuspended in 12.5 ml of freshly mixed and pre-chilled filtered solutions of 100 mM CaCl2 and 40 mM MgSO4, incubated on ice for 30 min, re-

centrifuged and finally ressuspended in 2.5 ml of 100 mM CaCl2 plus 2.5 ml of 40 mM

MgSO4. Pre-chilled glycerol was added to 10%, and the cell suspension was aliquoted

(200 µl per microfuge tube), frozen immediately in liquid nitrogen and stored at -70°C.