Retroviral Protocols

Retroviral vectors were created to express CEBPD and IK6. Variants of the Murine Stem Cell Virus (MSCV)-internal ribosomal entry site (IRES) retroviral plasmids (Figure 2.2) were combined with the RD114 envelope plasmid, which specifically targets HSCs and reduces cell toxicity (Bell et al., 2010), and the m57 gag/pol plasmid.

Figure 2.2. Retroviral Plasmids used during this study.

AMP = Ampicillin. Selection marker. Ori = Origin of amplification. Viral replication is initiated at this site. 5’/3’LTR = Long terminal repeats. Enhances transcriptional activation and prevents transcriptional repression, variable among viruses and

102

contain promoters, enhancers, and transcriptional termination. Ψ = Psi Packaging element. Regulates the packing of the retroviral genome in the viral capsid during replication. IRES = Internal ribosome entry site. Allows for the initiation of translocation in the middle of an mRNA sequence. Yellow boxes show target gene cDNA, green boxes represent the EGFP selection marker and orange boxes represent the Thy1 selection marker. MIGR1 denotes the MSCV-IRES-GFP empty vector plasmid, MiT denotes the MSCV-IRES-Thy1 empty vector plasmid (Coffin et al., 1997; Cherry et al., 2000; Buchschacher, 2001).

Retroviral particles were created using calcium phosphate co-precipitation and used to transduce CD34+ cells.

2.3.6.1 Calcium Phosphate Transfection

Calcium phosphate co-precipitation, introduced in 1973 (Graham and van der Eb, 1973) is a popular transfection method due to the low cost, ease of the technique, and efficiency with numerous culture and cell types. The technique functions by creating a precipitate of calcium, phosphate and DNA, which facilitates the binding of the precipitated DNA to the target cell surface, and entry into the cell via endocytosis. The protocol includes aeration of the phosphate buffer while adding the DNA-calcium chloride solution to ensure the resulting precipitate is fine, and not clumped, which hinders DNA adherence to the target cell surface. The protocol has several challenges including identifying and maintaining optimal pH for precipitate formation, causing cell toxicity due to buffer and salt concentrations, and a lower transfection efficiency in comparison to other chemical transfection methods.

Calcium Phosphate Transfection Protocol

2-3 million 293T cells were plated in a 10cm tissue culture treated plate 24 hours before transfection. Cells were transfected when ~70% confluent. All transfection reagents were thawed at room temperature and mixed thoroughly before use. Two tubes were prepared for the transfection, tube 1 containing DNA, water and CaCl2 in that order. 1ml of HBSS x2 was added to tube 2.

Reagents µg per dish

Viral envelope (RD114) 3

Viral gag/pol (M57) 10

103

Table 2.6. Insert DNA concentrations for retroviral particle synthesis per 10cm tissue culture dish.

In a tissue culture hood, while gently vortexing the HBSS x2 solution, the contents of tube 1 were added drop wise to tube 2, continuously vortexing. After combining, the mixture was left at room temperature for 30 minutes. After incubation the solution was once again vortexed and added drop wise to the 293T cells which were cultured in media appropriate for the cells to be transduced (Section 2.2.5.5). 10ul of 25mM chloroquine solution was also added. The plate was swirled to evenly distribute the virus. Plates were left for 24 hours before harvesting the first batch of virus.

2.3.6.2 Harvesting Viral Media

Viral medium was harvested from 293T cells 24 hours after transfection and replaced with fresh medium. Harvested media was syringe filtered (0.2µm), and was used fresh on cells undergoing transduction, remaining media was stored at -80°C. The second harvest was performed 12 hours after the first and followed the same protocol. The third and final harvest was performed 12 hours later.

2.3.6.3 CD34+ Cell Transduction

A 6 well tissue culture treated plate was coated with 2mls of retronectin, which was left either overnight at 4°C or for 1 hour at room temperature. Retronectin was collected and stored at -20°C to be re-used up to three times. The plate was washed with 1x PBS and blocked with 1x PBS + 2% BSA for 30 minutes at room temperature. The plate was washed with PBS and stored at 4°C with 2mls of PBS in coated wells to prevent them drying out. The plate can be stored for up to a week. Before use, PBS was aspirated and 3mls of viral media were added to coated wells, the plate was spun at 1000-2000 x g for 90 minutes. During this spin the cells for transduction were re-suspended in viral media with the appropriate cytokines and 4ug/ml of polybrene at a cell density of 125k cells per ml. The cells were centrifuged at 815 x g for 60 minutes. After spinning the cells were re-suspended in viral medium and cytokines and added to the retronectin coated wells. Cells were transduced twice more at 12 hour intervals. Medium was carefully aspirated and freshly harvested viral medium, cytokines and polybrene

104

were added. After a further 6-8 hours cells were harvested using disassociation buffer and plated out in normal medium with SCF, TPO, FLT3-L, all at 100ng/µl for up to 48 hours, to expand the primitive lymphoid population. Cells were plated out for specific experiments after 8 hours. No viral titrations were performed as the main aim of these experiments was the high expression of CEBPD and IK6. Transduced CD34+ cells were cultured in cytokine rich medium to stimulate expansion of haematopoietic cells and progenitors. Cytokines included in this study were as follows:

SCF – A growth factor with a broad range of activities including the promotion of haematopoiesis. SCF is expressed as a membrane bound protein and can also be cleaved into a soluble form, both forms promote proliferation. It performs most efficiently combined with other cytokines: such as TPO, GM-CSF, G-CSF, M-CSF, IL-3, and IL-7, to induce proliferation.

TPO - Thrombopoeitin is a regulatory factor for megakaryocytopoiesis and thrombopoiesis. It functions by stimulating the growth and maturation of

megakaryocytes, synergistically with other cytokines, to induce haematopoietic proliferation and differentiation.

FLT3-L – FLT3 Ligand binds and activates the FLT3 receptor. FLT3 is important in the development of dendritic cells. It synergistically stimulates the

proliferation and differentiation of haematopoietic cells and haematopoietic progenitors.

IL-3 - A growth factor which promotes survival, differentiation and proliferation of multiple myeloid and erythroid lineages. It also directly activates monocytes, suggesting additional immunoregulatory roles. This cytokine is heavily dependent upon co-stimulation of other cytokines.

IL-6 – A pleiotropic cytokine, which regulating immune and inflammatory responses. It stimulates B-cell differentiation and synergizes with IL-3 in megakaryocyte development and platelet production.

IL-7 – A hematopoietic growth factor, which stimulates the differentiation of pluripotent hematopoietic cells into lymphoid progenitor and lymphoid lineage (B cells, T cells and NK cells) cells.

105 2.3.6.4 Co-culture of CD34+ Cells

CD34+ transduced cells were cultured with MS-5 stromal cells in α-MEM medium with 20% FBS and 1% pen/strep and SCF, Flt-3L, IL-7 cytokines at 10ng/ml; 6 well tissue culture plates and 10cm tissue culture dishes were used for co-culture. Wells were coated with a 50% gelatin solution and left for one minute at room temperature, to encourage stromal cell adherence to the plastic surface. Stromal cells were plated out in the gelatin coated wells and grown to confluence. CD34+ cells were seeded at 80,000 cells per ml. Cells were monitored and split once or twice a week depending upon confluence. Fresh medium was added every time cells were split. When splitting cultures, the stromal layer was gently washed with medium to loosen settled cells while avoiding detaching the stroma, to provide an accurate representation of cells when performing cell counts and to harvest sufficient cells for flow cytometry analysis or cell collection.

2.3.6.5 Harvesting of Co-cultured CD34+ Cells for Flow Sorting

To harvest co-cultured CD34+ cells, first the medium was removed and stored to be spun down. Next, the well and stromal layer was washed several time with PBS and added to the collected media. 1X trypsin was added, covering the stromal layer, and incubated at 37°Cfor 3 minutes to loosen the cell layer. After the incubation step the stromal layer was carefully removed from the well and added to a separate tube, the well was washed with more PBS to collect any remaining cells and added to the medium tube. The falcon containing the stromal layer was vortexed to encourage disassociation of any remaining attached CD34+ cells. The stromal and medium were filtered through 50µl mesh to remove the larger stromal cells, then collated and spun down at 365 x g for 5 minutes. The resulting pellet was counted and stained following the FACS staining protocol (Section 2.3.9.3).