Gene transfer by retroviral transduction

2.2.2.1Vector construction

The complementary DNA (cDNA) of human FLT3-ITD (W51) was kindly provided by Prof. Dr.

med. Thomas Fischer and PD Dr. med. Florian Heidel (Magdeburg, Germany). W51 gave rise to duplication of human FLT3 amino acids 595–601 (REYEYDL).¹⁵ Self-inactivating retroviral vectors ‘pSRS11 SF iGFP pre’ was a gift from Prof. Dr. med Axel Schambach PhD (Hannover, Germany).¹¹⁰ Using Eco47III restriction enzyme site, we cloned human FLT3-ITD (W51) fragment into ‘pSRS11 SF iGFP pre’and generated ‘pSRS11 SF FLT3-ITD iGFP pre’. Based on

‘pSRS11 SF FLT3-ITD iGFP pre’, we generated human wild-type FLT3 and FLT3-ITD-N676K by overlap PCR.¹¹¹ Mutagenesis of N676K or D835Y ( hereafter FLT3-TKD835) was introduced into wt FLT3. PCR strategy and primer sequences were shown in figure 2.1 and table 2.1-2.2.

Materials and methods 32

Figure 2.1 Overlap PCR generate specific nucleotide mutations. Mutagenesis is introduced using mutagenic primers (b, c) and flanking primers (a, d). Adapted from Heckman et al, 2007.¹¹¹

Table 2.1 Strategies for construction of different FLT3 mutants by overlap PCR Constructs generated Constructs as

PCR templates

Restriction enzyme sites

Primer a

Primer b

Primer c

Primer d

FLT3-wt FLT3-ITD Muni+Agei KH35 KH42 KH43 KH41

FLT3-N676K FLT3-wt Muni+Agei KH35 KH57 KH58 KH41

FLT3-TKD835 FLT3-wt Muni+Agei KH35 KH36 KH37 KH41

FLT3-ITD-N676K FLT3-ITD Muni+Agei KH35 KH57 KH58 KH41

Table 2.2 Primers used for overlap PCR Primer name Sequences 5’-3’

KH35 CAT TCT ATG CAA CAA TTG GTG TTT GTC TCC TC

KH36 TCA TGA TAT ATC GAG CCA ATC CAA AGT CAC ATA TCT TC

KH37 TTG GCT CGA TAT ATC ATG AGT GAT TCC AAC TAT GTT G

KH41 ATT CAT GGT GGC GAC CGG TAG CAA CTA CGA ATC

KH42 CTT GGA AAC TCC CAT TTG AGA TCA TAT TCA TAT TC

KH43 GAA TAT GAA TAT GAT CTC AAA TGG GAG TTT CCA AG

KH57 GCA CGC CCC CAG CAG TTT CAC AAT ATT CTC GTG

KH58 CAC GAG AAT ATT GTG AAA CTG CTG GGG GCG TGC

KH68 GCG CTA CCG GTA TCC GCC CCT CTC

KH69 GGT TGT GGC CAT ATT ATC ATC GTG

KH70 AAT ATG GCC ACA ACC ATG CCG CGC GTC GTG CCC GA

KH71 GGG GGA TCC GTC TTA TTC ACT GGC CTT GGT TC

For ‘pSRS11 SF IRES dTomato pre’ construction, we replaced ‘IRES GFP’ of ‘pSRS11 SF iGFP pre’ with ‘IRES dTomato’ [ a gift from Franziska Geis/Prof. Dr. med Axel Schambach PhD (Hannover, Germany)] by using Agei and Noti restriction enzyme sites. Human CBFB-MYH11 cDNA¹⁰⁷ (provided by Prof. Dr. Christopher A. Klug, Birmingham, USA) was introduced into

‘pSRS11 SF IRES dTomato pre’ using Agei site. ‘pSRS11 SF CBFB-MYH11 IRES dTomato pre’ was generated.

To co-expressed FLT3-N676K and CBFB-MYH11 by single vector, ‘pSRS11 SF FLT3-N676K IRES CBFB-MYH11 pre’ was constructed. Firstly, we amplified ‘IRES CBFB-MYH11’ fragment by overlap PCR. Based on ‘pSRS11 SF CBFB-MYH11 IRES dTomato pre’ construct, primer KH68+KH69 and KH70+KH71 were used to amplify IRES and CBFB-MYH11,

Materials and methods 34

respectively. Overlap PCR product ‘IRES CBFB-MYH11’ was generated by primer KH68+KH71, introducing Agei+BamHi sites. This product was digested with Agei+BamHi and cloned into fragment of ‘pSRS11 SF FLT3-N676K IRES GFP pre’, in which ‘IRES GFP’ was removed by Agei+BamHi.

All constructs used in the present study were verified by sequencing.

(For schematic structure of constructs, see Figure 3.1A)

2.2.2.2 Plasmid production

Plasmids were transformed into competent XL1-Blue bacterial cells. Transfected cells were plated on LB-agar plates followed by incubation overnight at 37^oC. Subsequently, colonies were picked and proliferated at 37^oC in LB medium with appropriate agitation. Plasmids were purified by spin miniprep or maxi kits according to Qiagen’s manual. Dissolved in TE buffer, the plasmids were measured for concentration and stored at -20^oC until use.

2.2.2.3 Transfection of HEK293T cells by calcium phosphate

The mixture of plasmids was firstly prepared in distilled water containing calcium chloride (CaCl₂) as following: gene of interest containing retroviral vector, pcDNA3.MLV syn gag/pol [a vector for expression of optimized murine leukemia virus (MLV) matrix and core proteins, reverse transcriptase, RNase H and integrase), K73 eco-env (a vector for ecotropic envelope protein expression, recognizing a receptor only found in rat and mouse cells) (Table 2.3).

Next, 500µl plasmid mixture and 500µl HeBS solution (2X) were combined. The plasmid mixture was added in 1 drop every 2-3 seconds, while the solution was vigorously shaked by pipetting. The total mixture (1ml) was moved to a 60.1 cm² tissue culture dish containing 10ml

DMEM culture medium (with 25µM chloroquine) for culture of HEK293T cells. The cells were incubated at 37^oC for 8-10h. Afterwards, the medium was replaced by fresh DMEM culture medium with 20 mM HEPES.

Table 2.3 Components of transfection Plasmid and reagents Amount Retroviral vector 20µg

pcDNA3.MLV 10µg

K73 Eco 5µg

2.5M CaCl2 50µl

H2O ad 500µl

2.2.2.4 Virus collection and titration

At different time points (24h,36h,48h,60h,72h), the supernatants were collected, pooled, filtered with 0.22 µm filter and aliquoted for storage at -80^oC. Subsequently, the titer of virus was determined in SC-1 murine embryo fibroblast-like cells. 1x10⁵ SC-1 cells were cultured in 24-well plate in 100µl culture medium. Various amounts of virus (10µl, 25µl, 100µ l) were added into wells. Two days later, the transduction efficiency was measured by FACS according to GFP/dTomato/Gene of interest expression in transduced cells. Titer per ml=2x (transgene positive cells %) x (cell number at day of transduction)/volume of virus supernatant. The virus with high titer (>5x10⁵ TU/ml) was used for future experiments.

2.2.2.5 32D cell transduction

2 ml retronectin (48 µ g/ml) was added into the 6-well plate and incubated for 2h at room

Materials and methods 36

Washing by 3 ml HBSS/2.5% 1M HEPES was carried out before virus loading. Up to 1 ml virus supernatant was centrifuged under 2000 rpm, 4^oC for 30 min. Depending on different MOIs, more rounds of centrifugation could be performed for high transduction efficiency. (1-2)x10⁵ 32D cells were added into the well after removal of virus supernatant. Transgene expression was measured by FACS 2 days after transduction.