TRANSFEX - SUPERIOR GENE EXPRESSION FOR HARD-TO-TRANSFECT CELL TYPES. Kevin Grady Product Line Business Manager ASCB Vendor Showcase Dec.

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1 TRANSFEX - SUPERIOR GENE EXPRESSION FOR HARD-TO-TRANSFECT CELL TYPES Kevin Grady Product Line Business Manager ASCB Vendor Showcase Dec. 15, 2013

2 Outline Overview of transfection TransfeX Primary/hTERT cell transfection Stem cell transfection Continuous cell line transfection HEKPlus Protein Expression System 2

3 Mechanisms of transfection Physical Mechanical disruption of cell membrane opens pores in cell membrane Cell undergoes mitosis, plasmid DNA is captured by newly-formed nuclei of daughter cell Common forms: Electroporation and Microinjection Viral Recombinant virus is generated by a multi-step process including gene cloning, amplification in packaging cells, and high titer viral particle purification Infect cells (containing viral specific receptor) Common forms: Retrovirus (Lentivirus) and Adenovirus 3

4 Mechanisms of transfection Chemical DEAE-Dextran Calcium Phosphate Cationic lipid reagent Cationic non-lipid reagent Cationic Lipid Transfection Reagent N C DNA DNA and Cationic Lipids form a Complex DNA-Cationic Lipid Complex Interacts with the Mammalian Cell DNA is Expressed Post Transfection 4

5 Factors affecting transfection efficiency Cells Healthy and actively dividing Plating density and confluency at time of transfection Passage number DNA High purity and endotoxin free Choice of promoter Ratio of DNA versus lipid Amount of DNA and lipid Media Antibiotics Serum Polyanions 5

6 Factors affecting transfection efficiency DNA to Reagent Ratio Must mask negative DNA charge : Reagent Ratio Determined empirically Incubation Time Must balance penetration with viability Determined empirically 6

7 Outline Overview of transfection TransfeX Primary/hTERT cell transfection Stem cell transfection Continuous cell line transfection HEKPlus Protein Expression System 7

8 ATCC TransfeX transfection reagent (ATCC ACS-4005 ) TransfeX Reagent: Designed for transfection of primary cells, stem cells, and hard-to-transfect cell lines Validated in many ipscs, adult stem cells, primary cells, immortalized cell lines, and continuous cell lines Free from animal components Performance tested TransfeX DNA 15 minutes hours High efficiency transfection 8

9 Outline Overview of transfection TransfeX Primary/hTERT cell transfection Stem cell transfection Continuous cell line transfection HEKPlus Protein Expression System 9

10 Transfection of HUVECs with TransfeX and EF1α-GFP vector Phase GFP 48 hours 24 hours 71% GFP + 10

11 Transfection of HUVECs with alternate supplier reagent and EF1α-GFP vector Phase GFP 48 hours 24 hours 36% GFP + 11

12 Transfection of primary dermal microvascular endothelial cells with TransfeX and EF1α-GFP vector Phase GFP 48 hours 24 hours 75% GFP + 12

13 Transfection of immortalized human microvascular endothelial cells (TIME) with TransfeX and CMV-GFP vector Phase GFP 48 hours 24 hours 53% GFP + 13

14 Transfection of immortalized human microvascular endothelial cells (TIME) with alternate supplier reagent and CMV-GFP vector Phase GFP 48 hours 24 hours 14% GFP + 14

15 Outline Overview of transfection TransfeX Primary/hTERT cell transfection Stem cell transfection Continuous cell line transfection HEKPlus Protein Expression System 15

16 Transfection of BM-MSCs with TransfeX and EF1α-GFP vector Phase GFP 48 hours 24 hours 55% GFP + 16

17 Transfection of BM-MSCs with alternate supplier reagent and EF1α-GFP vector Phase GFP 48 hours 24 hours 16% GFP + 17

18 Transfection of hipscs with TransfeX and EF1α- GFP vector Phase GFP 48 hours 24 hours 90% GFP + 18

19 Outline Overview of transfection TransfeX Primary/hTERT cell transfection Stem cell transfection Continuous cell line transfection HEKPlus Protein Expression System 19

20 Transfection of Caco-2 cell line with TransfeX and EF1α-GFP vector Phase GFP 48 hours 24 hours 70% GFP + 20

21 Transfection of Caco-2 cell line with alternate supplier reagent Phase GFP Comp. 2 Comp. 1 4% GFP + 23% GFP + 21

22 Transfection of C2C12 cell line with TransfeX and EF1α-GFP vector Phase GFP 48 hours 24 hours 95% GFP + 22

23 ATCC TransfeX transfection guide Protocols for using TransfeX to transfect... Continuous LNCap MDA-MB-231 HepG2 Caco-2 C2C12 3T3-L1 NuLi-1 TIME RPTEC-hTERT htert-hme Stem Bone-marrow derived MSCs hipscs BT-142 Primary Dermal Fibroblasts Dermal Microvascular Endothelial Cells HUVECs RPTECs Large Airway Epithelial Cells hmecs Download this and our other free culture guides at Contact Technical Service at 23

24 Outline Overview of transfection TransfeX Primary/hTERT cell transfection Stem cell transfection Continuous cell line transfection HEKPlus Protein Expression System 24

25 HEKPlus Protein Expression System (ATCC ACS-4800-K ) A complete mammalian protein expression system using serum-free suspension cell cultures of HEK293 cells Component HEKPlus SF Suspension Cells HEKPlus SFM Medium HEKPlus Boost Solution GeneXPlus Transfection Reagent ATCC No. ACS-4500 ACS-4002 ACS-4003 ACS-4004 L-Alanyl-Glutamine, 200 mm

26 The kit components are optimized to work together Day 0 HEKPlus SFM + DNA + GeneX-Plus SEED AFTER 2-3 passages THAW 1. Count cells 2. Centrifuge 3. Aspirate supernatant 4. Resuspend pellet 0.8X10 6 cells/ml 24hrs. prior to transfection 3hrs Day 3-6 Shaker incubator 37 C and rpm Add 0.5% HEKPlus Boost soln. Return to the incubator Collect protein 26

27 HEKPlus GeneXPlus Transfection Reagent GeneXPlus Transfection Reagent (ACS-4004 ) 1 ml Animal component free Used for transfection of plasmid DNA into mammalian cells Formulated for low cytotoxicity; optimized to balance cytotoxicity and potency Produces high levels of gene expression Suitable for both transient and stable transfection 27

28 HEKPlus System: High transfection efficiency Cells expressing GFP (%) Phase Time (h) post transfection GFP The HEKPlus Expression system consistently achieves high transfection efficiency, with 65-70% of cells expressing the construct hours after transfection 28

29 HEKPlus System: Better yield versus an alternate supplier IgG1 Yield (µg/ml) ATIII Yield (µg/ml) HEKPlus Alternate supplier SEAP activity Day 6 (OD 640 nm/µl) HEKPlus Alternate supplier HEKPlus Alternate supplier HEKPlus system results in protein yields higher than the expression systems of an alternate supplier. SEAP was assayed using a phosphatase reaction, which suggests that the expressed protein is functional. 29

30 HEKPlus System: Cost-effective and scalable Kit components offered as a cost-effective complete system or individually to meet the needs of the investigator. The kit is scalable. It is tested to ensure a comparable yield of SEAP when either 2 ml or 200 ml of cells (1 x 10 6 cells/ml) are transfected. 30

31 Transfection of THP-1 cells with GeneX Plus and EF1α-GFP vector Phase GFP 24 hours 48 hours 38% GFP + 31

32 Transfection of Raw cells with GeneX Plus and EF1α-GFP vector Phase GFP 24 hours 48 hours 28% GFP + 32

33 Transfection of Raw cells with GeneX Plus and EF1α-GFP vector Phase GFP 24 hours 48 hours 40% GFP + 33

34 Summary: TransfeX and the HEKPlus Protein Expression System TransfeX Universal transfection reagent that can be used to transfect difficult-to-transfect cells like stem and primary cells High efficiency and low cytotoxicity Cost effective and scalable HEKPlus Serum-free, xeno-free fully optimized system simplifies purification and downstream processing. Efficient transfection reaction generates high-yield of functional protein. Kit is cost effective and scalable. GeneX Plus Reagent is also suitable for transfection of THP-1, Raw 264.7, SH-SY5Y, BM-MSCs, BJ-5ta, and HUVECs. 34