Transfection and genome engineering Innovation delivered selection guide 4 Contents Transfection decision tree 5 Superior transfection reagents 6

Lipofectamine® 3000 Transfection Reagent 6

Lipofectamine® RNAiMAX Transfection Reagent 10

Lipofectamine® MessengerMAX™ Transfection Reagent 11

ExpiFectamine™ 293 Transfection Kit 14 Broad-spectrum transfection reagent 15

Lipofectamine® 2000 Transfection Reagent 15

Electroporation 16

Neon® Transfection System 16 In vivo delivery 17

Invivofectamine 3.0 Reagent 17 Transfection-related products 19

Opti-MEM® I Reduced-Serum Medium 19

Selection antibiotics 19 Genome modulation and engineering 20

Ambion® siRNA 20

mirVana™ miRNA Mimics and Inhibitors 22

GeneArt® CRISPR tools 24

GeneArt® TAL effector tools 25 Ordering information 26 50,000 Transfection decision tree Transfection selection guide 45,000 Transfection Transfection 40,000 decision tree

35,000

Transfection is the process by which nucleic acids are introduced into eukaryotic cells. 30,000 PAYLOAD RNAi DNA mRNA Techniques vary widely and include lipid-based transfection and physical methods such as 25,000 ® siRNA, miRNA Cas9 DNA, GFP, Luc, shRNA Cas9 mRNA, GFP mRNA . Lipofectamine transfection reagents are the most trusted and cited in the 20,000

scientific literature due to their superior transfection performance and broad spectrum. 15,000

An overview of our most effective transfection products is shown in Table 1 to help you 10,000 COTRANSFECTION choose the solution that’s right for you. 5,000

0

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 201020112012 2013 2014

Table 1. Transfection selection guide—more blocks represent higher Cumulative number of publications citing the use of Lipofectamine® family of reagents since 1996. ® ® ® Lipofectamine® transfection efficiency into a greater number of cell types. Lipofectamine Lipofectamine Lipofectamine TRANSFECTION TM Difficult-to- REAGENT RNAiMAX 2000, 3000 3000 MessengerMAX Co- Easy-to-transfect transfect Primary Suspension Transfection product DNA mRNA RNAi delivery* cells cells cells Stem cells cells Superior transfection reagents Lipofectamine® 3000 Transfection Reagent Lipofectamine® RNAiMAX Transfection Reagent GREAT COULD BE POOR GREAT COULD BE POOR Lipofectamine® MessengerMAX™ RESULTS BETTER RESULTS RESULTS BETTER RESULTS Transfection Reagent ExpiFectamine™ 293 ™ Transfection Kit Recommended for Expi293F cells Broad-spectrum transfection reagent Lipofectamine® 2000 Transfection Reagent TRY TRY TRY TRY ® ® Electroporation 1 Reverse transfection 1 Neon Transfection 1 Different DNA 1 mRNA + Lipofectamine 2 Different cell density System concentration MessengerMAX™ reagent Neon® Transfection System 3 Increased siRNA 2 Viruses with shRNA 2 Different cell density 2 Plasmid DNA + Neon® concentration Transfection System In vivo delivery 3 mRNA + Neon® Transfection System Invivofectamine 3.0 Reagent In vivo delivery to liver following tail vein injection ® Neon 4 Viruses Transfection System

*Cotransfection of RNAi vector and siRNA.

4 5

Gentle with low toxicity Enhance your cancer research ® Superior transfection reagents Lipofectamine 3000 reagent was designed to optimize every step The superior transfection efficiency and broad cell spectrum of Superior in the transfection process. The superior transfection performance Lipofectamine® 3000 reagent enables excellent flexibility to use your

allows you to reduce the reagent dose and improve cell viability cell line of choice in cancer studies (Figure 4). transfection reagents ® Lipofectamine 3000 Transfection Reagent “We were very happy and surprised to see when working with your cell line of interest (Figure 3). ® Lipofectamine® 3000 reagent leverages our most advanced lipid Lipofectamine 3000 (reagent) provide a nanoparticle technology to enable superior transfection performance more than 10-fold difference in transfection and reproducible results. It delivers exceptional transfection efficiency efficiency in our difficult-to-transfect cell line. into the widest range of difficult-to-transfect and common cell types There was even a reduction in cell death. (Figures 1 and 2) with improved cell viability. Awesome results!” –Rui Eduardo Castro, PhD % Transfection University of Lisbon 100 Transfection ef ciency in cancer cell line panel Lipofectamine® 3000 reagent offers: 90 100 • Superior transfection efficiency—into the broadest spectrum of Lipofectamine® 3000

Lipofectamine® 3000 Lipofectamine® 2000 Competitor y 80 c difficult-to-transfect cell types ® n 80 Lipofectamine 2000 e i

70 c • Improved cell viability—gentle on your cells, with low toxicity f

e 60 HEK 293 60 on i 40 ec t

50 f s n a

40 r 20 T

HeLa % Transfection

30 % Lipofectamine® 2000 0 1 3 6 1 3 2 1 8 D - - - T L 2

® 2 R 20 s - 14 5 V 48 0 62 0 4 54 9 46 0 Lipofectamine 3000 46 8 H - o - L ak i - 578 T H O W a W T - E B - - C s ® ANC - S S S B DU - K M Lipofectamine LTX M P H 10 NC I - S HCC 193 7

LNCaP NC I K S DA -

0 M 0.1 0.2 0.3 0.4 Cell line Dose (µL) Western blot, HepG2 HepG2 Lipofectamine® Lipofectamine® Competitor Competitor 2000 3000 1 2 Figure 3. Superior transfection efficiency of Lipofectamine® 3000 Figure 4. Transfection efficiency in a cancer cell line panel. Lipofectamine® reagent at low doses. Each reagent was used to transfect HeLa cells in a 2000 reagent and Lipofectamine® 3000 reagent were used to transfect 17 cell GST-STAT 96-well format at the indicated doses with an emerald green fluorescent lines with a GFP-expressing plasmid in a 24-well plate format using 0.5 µg A549 protein (emGFP)–expressing vector. Analysis was performed 48 hours plasmid/well and the recommended protocols for each reagent. GFP expression posttransfection using flow cytometry to determine percent transfection was analyzed 48 hours posttransfection. Each condition was tested in triplicate, ® β-actin efficiency. Lipofectamine 3000 reagent delivered higher efficiency than both and the data points with error bars show mean transfection efficiency with Lipofectamine® 2000 reagent and Lipofectamine® LTX reagent. standard deviation. Figure 2. Lipofectamine® 3000 reagent outperforms Lipofectamine® 2000 and competitor reagents. Each reagent was used to transfect HEK 293, Figure 1. Enhanced protein expression using Lipofectamine® 3000 reagent. HeLa, LNCaP, HepG2, and A549 cell lines in a 96-well format, and green HepG2 cells were transfected with a vector expressing GST-tagged STAT fluorescent protein (GFP) expression was analyzed 48 hours posttransfection. protein. A western blot was performed to determine the level of expression using Lipofectamine® 3000 reagent provided higher GFP transfection efficiency than β-actin as a control. Lipofectamine® 2000 and competitor reagents for all five cell lines.

6 7

Generate induced pluripotent stem cells Improve editing outcomes Transfect stem cells ® ® Induced pluripotent stem cells (iPSCs) hold immense promise for Lipofectamine 3000 reagent was developed to break through Lipofectamine 3000 reagent was developed to unleash the power Superior the future of regenerative medicine and personalized therapeutic the boundaries of traditional delivery methods and facilitate new of stem cells by providing a highly efficient, cost-effective nucleic

treatments for a myriad of diseases and conditions. The superior technologies, such as genome engineering, in more biologically acid delivery alternative to electroporation (Figure 7). This advanced transfection reagents transfection efficiency of Lipofectamine® 3000 reagent in relevant systems. With this reagent, GeneArt® CRISPR vectors lipid nanoparticle technology minimizes the stress on cells caused conjunction with the Epi5™ Episomal iPSC Reprogramming Kit targeting the AAVS1 locus in HepG2 and U2OS cells show improved by electroporation, simplifies the reprogramming workflow, and enables highly efficient reprogramming of somatic cells without the transfection efficiency (Figure 6), mean fluorescence intensity, enables advanced gene-editing technologies. need for electroporation (Figure 5). and genomic cleavage. High transfection and genome editing efficiency is also observed with GeneArt® Precision TALs. These advancements in delivery help minimize painstaking downstream workflows, enable easier manipulation, and enhance site- specific insertion of into the genome. A B

A B

150,000 A B 200,000

150,000 100,000 intensity intensity P P 100,000 50,000 50,000 Mean OF Mean OF 0 0

DNA: 1.0 µg DNA: 1.3 µg Lipofectamine® 3000: 1.5 µL Lipofectamine® 3000: 1.5 µL SSEA4+/GFP+: 42% SSEA4+/GFP+: 69% 3 µL Lipofectamine ® 2000 1.5 µL Lipofectamine ® 3000 3 µL Lipofectamine ® 2000 1.5 µL Lipofectamine ® 3000 GFP MFI 247344 GFP MFI 456741

Figure 5. Reprogramming efficiency of Lipofectamine® 3000 reagent Figure 6. Transfection efficiency and protein expression using GeneArt® Figure 7. Transfection of stem cells. (A) H9 embryonic stem cells (ESCs) or (B) compared to electroporation. BJ fibroblasts as well as neonatal (HDFn) CRISPR Nuclease Vector. The vector contained an OFP reporter gene and was iPSCs were transfected using Lipofectamine® 3000 reagent. Cells were visualized and adult (HDFa) human dermal fibroblasts were reprogrammed to iPSCs by transfected with Lipofectamine® 2000 or Lipofectamine® 3000 reagent into (A) by fluorescence microscopy and processed using flow cytometry to determine transfection of Epi5™ vectors using either Lipofectamine® 3000 reagent or U2OS and (B) HepG2 cell lines. Bar graphs show reporter ; transfection efficiency. the Neon® Transfection System. Colonies were visualized by (A) brightfield images show fluorescence of corresponding cells expressing OFP. microscopy and (B) stained for alkaline phosphatase.

8 9

Lipofectamine® RNAiMAX Lipofectamine® MessengerMAX™ Transfection Reagent

Low cytotoxicity profile for Superior Transfection Reagent Lipofectamine® MessengerMAX™ mRNA Transfection Reagent Lipofectamine® RNAiMAX reagent offers an advanced, efficient easy optimization delivers amazing transfection efficiency in neurons and a broad solution for siRNA or miRNA delivery. No other transfection reagent Lipofectamine® RNAiMAX reagent enables maximal knockdown spectrum of primary cells, enabling improved application outcomes transfection reagents for RNA interference (RNAi) experiments provides such easy and and excellent cell viability across a 10-fold concentration range of and more biologically relevant research (Figure 9). That’s because “With Lipofectamine® MessengerMAX™ efficient delivery into a broad spectrum of cell types including the reagent. This makes Lipofectamine® RNAiMAX reagent easy to our novel lipid nanoparticle technology is optimized to deliver the primary cells, stem cells, and other hard-to-transfect cell types optimize for the lowest siRNA concentration that can be used while highest amount of mRNA possible without the nuclear entry step (reagent), I can transfect Cas9 and CRE (Figure 8). reducing cytotoxicity in your experimental system. Transfection- that is required with DNA. recombinase mRNA into preadipocytes and mediated cytotoxicity can mask the true phenotype of a target bone marrow–derived mesenchymal stem cells gene being studied, so minimizing the amount of reagent during Successfully transfect more predictive cell models with a reagent at high efficiency and without the toxic effects Superior transfection efficiency at low transfection is a critical factor for successful RNAi experiments. that provides: of viral vectors.” siRNA concentrations • Amazing transfection efficiency in neurons and primary cell types –Daniel Cohen, PhD When it comes to achieving effective gene knockdown, University of Pennsylvania • Faster protein expression with no risk of genomic integration Lipofectamine® RNAiMAX reagent easily outperforms other siRNA transfection reagents. High knockdown levels of target can • Up to 10x higher cleavage efficiency using CRISPR mRNA be achieved with as little as 1 nM siRNA.

Figure 8. Silencer® Select siRNA transfection with 100% Lipofectamine® RNAiMAX. Cells were transfected with Amazing transfection efficiency in neurons and primary cell types 90% ® ® 80% Lipofectamine RNAiMAX complexed with Silencer Select 70% siRNA at 30 nM/well. Knockdown of GAPDH mRNA was 60% assessed by qPCR. The cell density per well and amount 50% ® Neurons hNSCs BJ fibroblasts RAW 264.7 40% of Lipofectamine RNAiMAX used per well were as follows: 4 ® ™ 30% rat primary cortex cells: 10 cells/well, 0.6 µL; rat primary Figure 9. Lipofectamine MessengerMAX mRNA 20% hippocampus cells: 104 cells/well, 0.6 µL; rat glial precursor reagent outperforms a leading DNA delivery 10% 3 ® reagent and a leading mRNA delivery reagent in 0% cells: 8 x 10 cells/well, 0.4 µL; neuroblastoma-spinal cord Lipofectamine

I 4 I s 1 1 2 (NSC) cells: 1.2 x 10 cells/well, 0.3 µL; rat astrocyte cells: 1.2 ™ fresh isolated mouse cortical neurons, hNSCs, - ex 71 S 0 1 MessengerMAX t 4.7 sor - - F p u r C r 4 3 Average GAPDH mRNA remaining W NS C T C

T BJ fibroblasts, and RAW 264.7 cells. For all cells G

m x 10 cells/well, 0.3 µL; human astrocyte cells: 8 x 10 cells/ 2 6 RH 30 c o

P E mRNA reagent J A HUVE C

ec u ® ™ r W M except hNSCs, Lipofectamine MessengerMAX

oc a well, 0.3 µL; PC-12 cells (derived from a pheochromocytoma p ar y A

R 3

JN- DSRC T-1 reagent and the leading mRNA delivery reagent were li a l of the rat adrenal medulla): 8 x 10 cells/well, 0.3 µL; hi p

Pri m Rat astrocytes G 4

Negative control megakaryoblastic leukemia cells (MEG-01): 2 x 10 cells/well, used to deliver GFP mRNA (500 ng/well) in a ar y Human astrocytes

m 4 i 24-well format. For all cells except hNSCs, the r 0.3 µL; murine dendritic cells (JAWSII): 1.2 x 10 cells/well, P 0.3 µL; human erythroleukemia cells (TF-1): 104 cells/well, 0.3 leading DNA delivery reagent was used to deliver Leading reagent Neuronal and glial precursor Blood cell Soft tissue cell µL; RAW 264.7 macrophages: 104 cells/well, 0.3 µL; human GFP DNA (500 ng/well), and GFP expression was umbilical vein endothelial cells (HUVEC): 8 x 103 cells/well, 0.3 DNA analyzed 24 hours posttransfection. For hNSCs, µL; rhabdomyosarcoma cells (RH30): 8 x 103 cells/well, 0.3 Lipofectamine® MessengerMAX™ reagent and the µL; human desmoplastic small round cell tumor cells (JN- leading mRNA delivery reagent were used to deliver DSRCT-1): 8 x 103 cells/well, 0.3 µL; human Ewing’s sarcoma GFP mRNA (250 ng/well) in a 48-well format. The cells (TC71): 1.6 x 104 cells/well, 0.3 µL. leading DNA delivery reagent was used to deliver Leading reagent GFP DNA (250 ng/well), and GFP expression was mRNA analyzed 24 hours posttransfection. To learn more, go to lifetechnologies.com/rnaimax

10 11

Faster protein expression with no risk of genomic integration Getting started is as easy as 1-2-3! ® TM Transfection of mRNA with Lipofectamine MessengerMAX reagent results in faster protein expression because translation of mRNA occurs in the Superior cytoplasm. Additionally, delivery of mRNA does not require nuclear entry (Figure 10, step 4), which eliminates the risk of genomic integration, and

transfection efficiency becomes cell cycle–independent. transfection reagents

1 2 3 1. 2. 3. 4. Protein Attachment Endocytosis Escape Nuclear expression Prepare DNA template Generate mRNA Transfect mRNA Prepare your DNA template with a T7 promoter. Transcribe your template DNA with the mMESSAGE mMACHINE® Transfect your mRNA with our simple to cell into cell from endosome entry You may choose to clone your gene with a T7 ULTRA Kit to generate mRNA for transfection. Lipofectamine® MessengerMAX™ Gateway® pcDNA™-DEST40 Vector or amplify transfection protocol. it with PCR and T7-containing primers.

Cloning option Reagent– T7 attR1 CmR ccdB attR2 V5 epitope 6xHis Timeline Steps DNA complex Seed cells to be 70–90% BGH p 1 confluent at transfection A Day 0 MV f C 1 o P ri DNA Diluted MessengerMAX™ Reagent ™ Dilute MessengerMAX reagent S V ® 4 2 in Opti-MEM medium (2 tubes) 0 —mix well o Vortex 2–3 sec r i Diluted mRNA

Nucleus A ® Prepare diluted mRNA master

m Gateway

™ p DNA transfection n 3 mix by adding mRNA to Endosome pcDNA -DEST40 Vector i i ® c c Opti-MEM medium—mix well i y l l in m R o Expression e Add diluted mRNA to each tube N

Day 1 ™ vector DNA 4 of diluted MessengerMAX reagent (1:1 ratio) A p mRNA transfection 40 p SV UC ori ARCA-capped mRNA 5 Incubate Protein with poly(A) tail

6 Add mRNA-lipid complex to cells

Visualize/analyze transfected 7 cells

PCR option Day 2–4 mRNA T7 ORF Reagent– mRNA complex Figure 11. mRNA transfection workflow.

Figure 10. DNA vs. mRNA transfection.

To learn more, go to lifetechnologies.com/messengermax

12 13 ExpiFectamine™ 293 Transfection Kit The ExpiFectamine™ 293 Transfection Kit is a core component of the Broad-spectrum transfection reagent Expi293™ Expression System. It is designed for transient transfection of high-density cultures of human embryonic kidney (HEK) 293 ® cells. The high-efficiency, cationic, lipid-based transfection reagent Lipofectamine 2000 Transfection Reagent and transfection enhancers are designed to power the highest Lipofectamine® 2000 reagent is the most referenced transfection 50,000 ™ ® possible level of protein expression from Expi293F Cells cultured in reagent in the scientific literature (Figure 13). Lipofectamine 2000 45,000 Expi293™ Expression Medium. reagent’s success can be attributed to its reliable performance, delivering a high level of transfection efficiency for DNA, RNA, 40,000 The ExpiFectamine™ 293 Transfection Kit: or cotransfection in a broad range of common cell types. The 35,000 Broad-spectrum

• Is designed specifically for transfection of high-density reagent offers: transfection reagent suspension cells in culture, with enhancers that boost transfection 30,000 • Reliable performance—high transfection efficiency in a broad range performance and protein expression of common cell types 25,000 • Achieves protein yields 2- to 10-fold higher than other transfection • Versatility —single reagent for DNA, RNA, and cotransfection 20,000 reagents that are used with high-density HEK 293 cell cultures (Figure 12) • Proven efficacy in the presence of serum—eliminates the need to 15,000 change media following transfection • Employs the same transient expression protocols typically used in 10,000 current low-density HEK 293 suspension culture systems, allowing 5,000 you to to easily switch from low-density systems to the Expi293™ Human lgG Cripto 0 Expression System 1,200 1,200 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 201020112012 2013 2014 • Provides robust and reproducible transfection results, giving you 1,000 1,000 greater confidence in results Figure 13. Cumulative number of publications citing the use of 800 800 ® • Allows you to scale for culture volumes less than Lipofectamine family of reagents since 1996. 1 mL to greater than 10 L while maintaining equivalent volumetric 600 600 protein yields 400 400

hIgG (µg/mL)

Cripto (µg/mL) 200 200

“In all my years working with transient 0 0 expression systems, the Expi293TM 30 mL culture 30 mL culture expression system is the first one to FreeStyle™ 293 Expi293™ achieve 2.3 grams/L beating every other Figure 12. Expression of human IgG and Fc-tagged Cripto protein achieve expression HEK293 transient expression system." levels of over 1 g/L in the Expi293™ Expression System. –Jelte-Jan Reitsma Research associate

To learn more, go to lifetechnologies.com/expi293 To learn more, go to lifetechnologies.com/2000

14 15 The Neon® Transfection System Superior transfection effi ciency for labs of all sizes, budgets, and needs Electroporation In vivo delivery Complexation buffer siRNA duplex 1 Protein analysis The Neon® Transfection System is a next-generation ® Neonelectroporation Transfection device System for highly effi cient transfection Invivofectamine 3.0 Reagent ® The Neon® Transfection System is a next-generation electroporation Neon Transfection System Invivofectamine® 3.0 Reagent is a breakthrough reagent for in vivo RNAi delivery, with Invivofectamine 3.0 Diluted siRNA of primary cells, stem cells, and diffi cult-to-transfect Reagent device for highly efficient transfection of primary cells, stem cells, Superior transfection efficiency for labs of all sizes, budgets, and needs greatly improved performance and up to 85% knockdown achieved using microgram 2 and othercells. difficult-to-transfect Find out how cellsresearchers (Figure 14). areThe benefiflexible tting and from the • Maximum efficiency—up to 90% in many cell types, including difficult-to-transfect levels of siRNA. open systemfl exibility, allows yousimplicity, to perform and high-quality versatility transfections of the Neon using® Trans- cells, primary, and stem cells 4 6 • Easy to use—siRNA complexes are ready to deliver in just a few steps optimized or user-defined protocols in three simple steps with as few • Flexible—easily transfect from 1 x 10 cells to 5 x 10 cells per reaction with easy- fection System. to-use protocols as 2 × 104 cells per reaction. A novel reaction chamber provides a • Effective, targeted knockdown—up to 85% knockdown observed with targets tested • Simple—easy to use, with single reagent kit for all cell types 3 dramatic increase in transfection efficiency and cell viability. • Use less siRNA sample—up to 90% less siRNA than required with previous reagent • Versatile—open system allows electroporation parameters to be optimized freely Complex for effective knockdown • Effi ciency—up to 90% in many cell types, including Learn more about the advantages of the Neon® Transfection System The Neon® Transfection System is: diffi cult-to-transfect cells, primary, and stem cells at lifetechnologies.com/neon. • Sustained knockdown—prolonged knockdown observed following a single injection 50ºC 30 min • Efficient—up to 90% efficiency in many cell types, including • Low toxicity—complexes exhibit extremely low in vivo toxicity difficult-to-transfect• Flexibility —easilycells, primary transfect cells, and from stem 2 xcells 104 cells to 6 x 106 4 A B • Flexible —easilycells per transfect reaction from 2 × 104 cells to 6 × 106 cells Easy to use Electroporation per reaction Creating complexes of Invivofectamine® 3.0 Reagent and RNAi duplexes for delivery is • Simplicity—single reagent kit for all cell types easy: simply mix, incubate, dilute, and inject (Figure 1). • Simple—easy to use, with a single kit that includes reagents for all Complex cell types• Versatility—open system allows electroporation Effective, targeted knockdown Lateral • Versatile parameters—open system to allows be optimized electroporation freely parameters to be ® ® caudal vein 5

Complexes of Invivofectamine 3.0 Reagent and Ambion in vivo siRNA targeting Factor delivery optimized freely VII or Stealth™ RNAi PPIB have been successfully delivered by mouse tail vein injection to High transfection efficiency of Jurkat cells with the Neon® Tranfection System. Jurkat cells were transfected ®

® liver tissue (Figure 2). We have demonstrated effective knockdown of Factor VII and PPIB In vivo withHighFigure an EGFP-encodingtransfection 14. High transfection vector effi usingciency the Neonefficiencyof Jurkat® Transfection cells of JurkatSystem with andNeoncells viewed Tranfectionwith using the (A) bright-fieldNeon System. microscopy Dorsal vein “It's actually helped me to add some assays that previously I had andIntracelluarTransfection (B) fluorescence uptake System. microscopy of Cellsreporter 24 hrwere following vector analyzed transfection. encoded by (A) with brightfield EGFP at and 24(B) hr fluorescencefollowing at the mRNA level. microscopy for intracellular uptake of enhanced GFP (EGFP) reporter vector 24 “I wouldto abandon recommend because it simply of low because transfection it was effi ciency.” transfection of Jurkat cells with Neon® Transfection System. (B) is the hours following transfection. very—Alina easy to Mares use, it was actually a little bit corresponding fl uorescence image of (A). 120% cheaper than the system we've used in the Lipofectamine® LTX and PLUS™ Reagent 100% !"#$%&'!()*)*+% Figure 1. An injection-ready RNAi/Invivofectamine® past and…I really like the features such as %!!"!!#$ Efficient reagent for plasmid delivery and protein expression -!"!!#$ 3.0 Reagent complex can be prepared quickly and in “I would recommend it simply because it was very easy to use, and RNAi Transfection it See Claire Demenis discuss the ,!"!!#$ 80% +!"!!#$ the database.” ® just a few steps. • Superior performance—high transfectionbenefi efficiency ts of the Neonwith maximal transfection (>90%) systemcell *!"!!#$ was actually a little less expensive than the system we've used )!"!!#$ ® —Lydia Wunderley, PhD =3.7$>/=?6@6@0$ Figure 2. Use of Invivofectamine 3.0 Reagent viability at lifetechnologies.com/neon 60% (!"!!#$ '!"!!#$ enables targeted knockdown in the liver after a &!"!!#$ Universityin the past of Manchester and I really like the features such as the database.” Easy—simple protocol provided for many cell lines ® • %!"!!#$ single intravenous injection. Invivofectamine 3.0 40% !"!!#$ • Versatile—superior plasmid delivery into a broad range of cell types including ./0$1234$563.7$ 889:$563.7$ ;<99$563.7$ —Dr. Lydia Wunderley expression % remaining Reagent complexed with siRNA targeting mRNA for primary and hard-to-transfect cells Factor VII (FVII) or PPIB, injected at doses of 1 mg 20% mRN A per kilogram mouse body weight (mg/kg), achieved Learn more about the advantages of Lipofectamine® “It’s very cost effi cient because you get high-effi ciency LTX Reagent at as much as 85% knockdown of target mRNA levels 0% lifetechnologies.com/ltx. ® throughput. The transfection is always successful and that saves No Treat. Neg CTRL PPIB siRNA FVII siRNA (knockdown assessed via TaqMan Assay). To learn more, go to lifetechnologies.com/neon siRNA you time and effort elsewhere [so] you can trust this one.”

16 —Claire Demenis 17 Product Cat. No. “The speed that you can actually [transfect] in comparison to the Neon® Transfection System MPK5000 reagent-based transfection system is a lot faster. You can do it in Neon® Transfection System Starter Pack MPK5000S fi ve minutes, instead of an hour or so.” 0 ng 500 ng 1,000 ng —Mathlew Hayley Lipofectamine® LTX Reagent with PLUS™ Reagent, efficiently transfects primary neural progenitor cells. Lipofectamine® LTX Reagent (1.5 μL) was used to transfect murine embryonic primary neural progenitor cells with the indicated quantities of a GFP-expressing plasmid in the presence of PLUS™ Reagent (2.5 μL). GFP expression was analyzed 24 hr post-transfection. Data courtesy of the Beverly Davidson laboratory, University of Iowa. To learn more and request a demo, go to lifetechnologies.com/neon

For research use only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affi liate(s) or their respective owners. CO04537 0113

124 ©2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Less siRNA required to achieve effective knockdown Complexes of Invivofectamine® 3.0 Reagent and siRNA in a range of amounts were introduced Transfection-related products via tail vein injection. FVII protein levels in the serum were measured using a chromogenic assay 24 hours after injection (Figure 3). The amount of knockdown is correlated with the amount of Opti-MEM® I Reduced-Serum Medium Selection antibiotics siRNA in the complex. The ED50 of Invivofectamine® 3.0 Reagent is 0.1 mg/kg, compared to ® ® previous levels of 1.0 mg/kg. Opti-MEM I Reduced-Serum Medium is a modification of Eagle's Gibco high-quality selection agents provide unique solutions for Minimal Essential Medium, buffered with HEPES and sodium your research needs, such as dual selection and rapid generation of Low in vivo toxicity bicarbonate, and supplemented with hypoxanthine, thymidine, stable cell lines (Table 2). To evaluate in vivo toxicity of Invivofectamine® 3.0 Reagent, several detailed studies focusing sodium pyruvate, L-glutamine, trace elements, and growth on analysis of a blood chemistry panel, hematology, and cytokine levels at multiple time points factors. Most cells grown in serum-supplemented medium can be ® post-injection were performed (Figure 4). The results show that the levels of various biomarkers in transferred to Opti-MEM medium with a minimum of 50% reduction ® mice transfected using Invivofectamine® 3.0 Reagent were not significantly different from those of in serum. We recommend Opti-MEM medium for dilution of untreated mice. transfection reagent and nucleic acids prior to complex formation.

1,000

Invivofectamine® 3.0 To learn more, go to lifetechnologies.com/optimem 90% !"##$%&'(#$ 80% 100 Invivofectamine® 3.0 70%

60% Table 2. Eukaryotic selection antibodies. 50% 10

Units Most common Common working remaining % 40% Selection antibiotic Available powder sizes Available liquid sizes selection usage concentration 30% mRN A 20% 1 Blasticidin Eukaryotic and 1–20 µg/mL 50 mg 10 x 1 mL, 20 mL bacterial cells 10% Geneticin (G418) Eukaryotic cells 100–200 µg/mL (bacteria) 1, 5, 10, or 25 g 20 mL, 100 mL 0% GLU ALP ALT AST TBIL CHOL TRIG 0.01 0.1 1 10 0.1 200–500 µg/mL (mammalian FVII siRNA mg/kg 1 3 U 1 3 U 1 3 U 1 3 U 1 3 U 1 3 U 1 3 U cells) Blood chemistry markers Hygromycin B Eukaryotic cells and dual- 200–500 µg/mL – 20 mL selection experiments Figure 3. Invivofectamine® 3.0 Reagent and siRNA Figure 4. Evaluating in vivo toxicity markers over time and with varying reagent doses. Invivofectamine® 3.0 targeting FVII produce dose-response knockdown Reagent complexed with Ambion® in vivo siRNA targeting FVII was injected into mice at doses of 1 and 3 mg/kg. Mycophenolic acid Mammalian and bacterial 25 µg/mL 500 mg – in liver after a single intravenous injection. Blood samples were collected at 2, 24, and 48 hr and were evaluated using clinical chemistry assays for several cells products ® ® Invivofectamine 3.0 Reagent complexed with Ambion biomarkers (Antech). Puromycin Eukaryotic and 0.2–5 µg/mL – 10 x 1 mL, 20 mL in vivo siRNA targeting FVII was injected at doses bacterial cells Transfection-related ranging from 0.02 to 2 mg/kg. Blood serum was isolated and assayed for FVII protein levels (Biophen® Zeocin Mammalian, insect, yeast, 50–400 µg/mL – 8 x 1.25 mL, 50 mL chromogenic assay). bacterial, and plant cells

To learn more, go to lifetechnologies.com/invivofectamine To learn more, go to lifetechnologies.com/selectionantibiotics

18 19 Genome modulation

20 and engineering

Genome modulation and engineering and modulation Genome † * • prediction to algorithms,effect chemistry and offer: incorporateand the latest improvements off-target design, insiRNA interfering (siRNAs). RNAs interfering Silencer unmodified 21-mer together annealed small to oligonucleotides form involvedcells the of use synthetic duplexes RNA of consisting two Traditionaltypes. knockdown methods inmammalian for gene to expression studyproteingene of range cell inawide function way down isthe best knock to (RNAi) interference effectively RNA Ambion been validatedbeen with Lipofectamine • • • Scientific. For more information, go to lifetechnologies.com Fisher from available not currently are products engineering and modulation Gene extend to any replacement product. replacement to any extend Silencer new to two up of replacement aone-time successful, is control positive appropriate an and observed not is knockdown of level guaranteed the If efficiency. transfection demonstrate to siRNA control apositive with knockdown >80% show also must Customers application. this for required not but recommended is PCR Real-time posttransfection. hours 48 detected levels mRNA and ≥5 nM at transfected been have must siRNAs guarantee, the for To by 70% more. or mRNA qualify target the silence will siRNAs two those target, same We guarantee that when you purchase two Silencer two purchase you when that We guarantee Silencer potencyHigh PubChem from our Silencer access Open in your reagents confidence results Guaranteed modifications off-targetreduce up by 90%effects to Minimal off-target effects off-target Minimal ® Select siRNA designs will be provided free of charge. This guarantee does not not does guarantee This charge. of free provided be will designs siRNA Select ® siRNA (Figure 17, siRNA Table 3) ® siRNA —65,000 siRNA sequences and associated data on and —65,000 sequences siRNA —improved to accuracycompared prediction siRNA —100% guaranteed† to for increased silence —locked (LNA) chemical —locked chemical (LNA) acid nucleic ® Select siRNA library siRNA Select ® ® RNAiMAX Transfection Reagent RNAiMAX Select siRNA products siRNA Select have ® Select Predesigned siRNAs to the effective siRNA prediction accuracy. prediction siRNA effective The Figure 17.Figure Silencer elicited that siRNAs of percentage the shows inset The cells. No. 1siRNA–treated real-time PCR using TaqMan using PCR real-time by posttransfection hours 48 measured was knockdown mRNA cells. HeLa in nM 5 at algorithm previous the using designed siRNAs with by side side tested were siRNAs These targets. to different 40 155 siRNAs to design used was algorithm as percent of mRNA remaining compared to Silencer compared remaining mRNA of percent as % mRNA remaining 1 0 20 30 40 50 60 70 80 90 1 0 0 0

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1 51 enhanced stability and safety profile safety studies. and animal for stability enhanced potency, including siRNAs specificity, and efficacy, an as as well 3.0 retain the and of all characteristics Silencer Reagent Ambion To more, learn go to lifetechnologies.com/sirna resitant Ambion use necessitate the RNase- of challenges added These fluids. body inall to present due levels the high counterparts of ribonucleases vivo In A solution vivo for successful in Table Ambion 3. Target species Coverage Target specificity Efficacy (>70%knockdown) Potency in vitro in their than are more challenging experiments RNAi ® In Vivo In ® ® siRNAs have validated siRNAs been with Invivofectamine siRNA selection guide. selection siRNA In Vivo In siRNAs to siRNAs results. obtain reproducible custom toolforotherspecies) Human, mouse,rat(use Coding RNA Moderate 2 of3siRNAguaranteed Silencer 100 nMrecommended conc. Cost-effective siRNA ® siRNA RNAi ® Select Select

custom toolforotherspecies) Human, mouse,rat(use Coding RNA High 2 of3siRNAguaranteed 20 nMrecommended conc. Stealth RNAi low off-target effects Good knockdown, please contact [email protected] targets, are backed and by a100% guarantee. For more information, custom be predefined can or sets made major to libraries gene of inhuman, mouse, experiments systems. rat and cell RNAi siRNA for performing siRNAs We of provide quality premium collections Ambion ™ siRNA ® siRNA libraries custom toolforotherspecies) Human, mouse,rat(use Coding andnoncodingRNA Highest 2 ofsiRNAguaranteed 5 nMrecommended conc. Silencer off-target effects Highest knockdown,lowest ® SelectsiRNA 21 mirVana™ miRNA Mimics and Inhibitors Genome editing ™ 1.40 mirVana miRNA Mimics and Inhibitors are chemically modified, FW Engineering made easy RV synthetic nucleic acids designed to either mimic mature miRNAs, 1.20 With the wealth of data available through advanced sequencing or to bind to and inhibit endogeneous miRNAs. These products 1.00 technologies, the next challenge for the research community is to provide a means to functionally study the role of specific miRNAs apply this information for more valuable, clinically relevant output. Chromosome & DNA-speci c within cellular systems, or to validate the role of miRNAs in regulating 0.80 The ability to edit the genome in a precise and targeted manner Engineered Nuclease double-stranded break ™ target genes. mirVana miRNA Mimics and Inhibitors have been can be used to provide a more comprehensive understanding of validated with Lipofectamine® RNAiMAX Transfection Reagent for 0.60 Fold change and disease mechanisms. Advances in use in cell-based systems, and with Invivofectamine 3.0 Reagent for 0.40 tools have enabled scientists to modulate or edit genes with simple Gene in vivo delivery. In vivo–ready mirVana™ miRNA Mimics and Inhibitors Gene yet powerful genome editing techniques (Figure 19). Genome editing Disruption Insertion Gene Gene 0.20 Addition Correction have been purified by HPLC and dialysis, making them ready for has a variety of applications such as creating disease-resistant immediate use. 0.00 transgenic plants, stem cell engineering and , and is miRNA Neg miRNA Neg miRNA Neg miRNA Neg miRNA Neg miRNA Neg Gene mirVana™ miRNA Mimics and Inhibitors are: mimic mimic mimic mimic mimic mimic also widely used in creating tissue and animal disease models. Deletion Inversion HOMOLOGY-DIRECTED REPAIR (HR) reporter 1 reporter 2 reporter 3 reporter 4 reporter 5 reporter 6 With added homologous DNA: • Versatile—functionally study specific miRNAs using in vitro or insert/replace DNA in vivo systems Figure 18. The mature strand of mirVana™ miRNA Mimics are highly potent. NON-HOMOLOGOUS END JOINING (NHEJ) miRNA mimics, like natural miRNAs, have 2 strands—the mature strand (guide Without added homologous DNA: • Potent—validate miRNA regulation of gene expression with repairs with indels strand) that is functional and used by Ago protein to target mRNAs, and the minimal off-target effects (Figure 18) star or passenger strand that is nonfunctional and is normally cleaved and • High-throughput compatible—generate libraries for effective expelled from the complex. Most scientists want to analyze one strand of miRNA Figure 19. Engineered nuclease-mediated genome editing. The CRISPR-Cas9 at a time, and want the other strand to be totally inactive. A key advantage of system or TAL effector nucleases can be designed to target specific sites in screening of multiple miRNAs simultaneously ™ mirVana miRNA Mimics is inactivation of the star strand. For this assay we the genome, creating double-strand breaks (DSBs) at desired locations. The • Current —content is regularly updated based on the miRBase measured activity from both strands of miRNA mimics. One reporter has a target natural repair mechanisms of the cell repair the break by either homologous in forward orientation to measure activity of the mature miRNA strand, and the miRNA database recombination (HR) or non-homologous end joining (NHEJ). HR is more precise, other reporter has the target cloned in reverse/complement orientation to test since it requires a template, allowing the introduction of foreign DNA into the activity of the star strand of the miRNA mimic. For all 6 sequences, activity of the target gene. Homologous DNA “donor sequences” can be used with homology- mature strand is high (5-10–fold knockdown compared to negative control), and directed repair (HDR) to introduce a defined new DNA sequence. DSB repair by ™ activity of the star strand is low (similar to negative control). NHEJ is likely to introduce errors such as insertions or deletions (indels), leading mirVana miRNA Libraries to a non-functional gene. Complete mirVana™ libraries containing mimics and inhibitors for every human, mouse, and rat miRNA are available. For information on all our predefined and custom miRNAs libraries, contact us at [email protected] and engineeringand Genome modulation

To learn more, go to lifetechnologies.com/mirna

22 23 ® GeneArt® CRISPR U6 IVT gRNA (from GeneArt® ® ® GeneArt CRISPR tools GeneArt GeneArt® Strings™ DNA CRISPR T7 Strings™ DNA) GeneArt TAL effector tools CRISPR CRISPR Rapid and efficient editing with multiplexing capabilities vector with vector with Precise and flexible editing with freedom to innovate OFP reporter CD4 reporter The CRISPR-Cas9 system is a new approach to genome editing TAL effectors are a widely used technology for precise and efficient that provides rapid, efficient editing with multiplexing capabilities. Cas9 mRNA Cas9 mRNA gene editing. The deciphering of the TAL effector “code” led to With their highly flexible but specific targeting, CRISPR-Cas9 the engineering of designer TAL effector proteins. GeneArt® TALs systems can be manipulated and redirected to become powerful provide custom DNA binding proteins engineered and designed tools for genome editing. The endonuclease cleavage specificity in for accurate DNA targeting and precise genome editing. GeneArt® Cas9 CRISPR-Cas9 systems is guided by RNA sequences, so editing can TALs have been validated with Lipofectamine® 3000 (Figure 22) be directed to virtually any genomic locus by engineering the guide NHEJ and offer site-specific delivery of nucleases, activators, repressors, RNA sequence and delivering it along with the Cas9 endonuclease (non-homologous end joining) chromatin modifiers, genomic labels, and crosslinking molecules HDR (homology-directed repair) to your target cell. Based on your research needs, you can Cotransfect cells with donor DNA (Figure 23). Based on your research needs you can choose from our choose from our two different formats of CRISPR tools: GeneArt® two different formats of TAL effector tools: GeneArt® Precision TALs CRISPR Nuclease all-in-one expression when working when you are working with plants or have no design constraints, or with mammalian systems, or the GeneArt® CRISPR Nuclease GeneArt® PerfectMatch TALs that do not require a 5’ T for complete mRNA System for improved editing efficiencies and multiplexing Gene disruption—repair to design flexibility. capabilities (Figures 20 and 21). native sequence results in DNA insertion—insert promoter, frameshifts or mutations Gene correction gene tags, and single or multiple genes GeneArt® CRISPR In vitro transcribe gRNAs bp Nuclease mRNA Use MEGAshortscipt™ Figure 20. Available GeneArt® CRISPR-Cas9 genome editing tools. The T7 Transcription Kit CRISPR-Cas9 system is composed of a short noncoding guide RNA (gRNA) 500 for gRNA synthesis that has two molecular components, a target-specific CRISPR RNA (crRNA), 400 300 IVT gRNAs and an auxiliary trans-activating crRNA (tracrRNA). The gRNA unit guides the Cas9 protein to a specific genomic locus via base pairing between the crRNA VP16/KRAB 200 TRANSFECT sequence and the target sequence. Upon binding to the target sequence, the with Lipofectamine® Cas9 protein induces a double-strand break at the specific target sequence. ™ MessengerMAX reagent Following CRISPR-Cas9–induced DNA cleavage, the break can be repaired by 100 the cellular repair machinery using either non-homologous end joining (NHEJ) or a homology-directed repair (HDR) mechanism. 20

40 15 Cas9 mRNA + IVT gRNA ® 35 All-in-one CRISPR plasmid Figure 21. Cleavage efficiency of various GeneArt CRISPR formats targeting 10 ® ®

the HPRT locus in Gibco iPS cells in a 12-well format. Lipofectamine Cleavage ef ciency 30 3000 reagent was used to deliver CRISPR nuclease all-in-one plasmid DNA; 5 ® ™ 25 Lipofectamine MessengerMAX and two leading mRNA delivery reagents were used to deliver an all-RNA CRISPR format (Cas9 mRNA + IVT gRNA). Cleavage 0 20 ® ® ® efficiency was determined using the GeneArt Genomic Cleavage Detection Kit Lipofectamine Lipofectamine engineeringand

2000 3000 Genome modulation 15 72 hours posttransfection. Figure 23. Available TAL effector domains. Cleavage ef ciency (%) 10 Figure 22. Cleavage efficiency for GeneArt® TALs. TALs targeting the AAVS1 locus were transfected by Lipofectamine® 2000 or Lipofectamine® 3000 reagent 5 in the U2OS cell line. Cleavage was assayed using the GeneArt® Genomic 0 Cleavage Detection Kit. Lipofectamine® Competitor A Competitor B Lipofectamine® 3000 Cells only MessengerMAX™ reagent To learn more, go to lifetechnologies.com/CRISPR To learn more, go to lifetechnologies.com/tals

24 25 Ordering information

Superior transfection reagents Quantity Cat. No. Electroporation continued Quantity Cat. No. Lipofectamine® 3000 Transfection Reagent 0.1 mL L3000001 Neon® Transfection System 100 µL Kit 96 x 2 reactions MPK10096 Lipofectamine® 3000 Transfection Reagent 0.75 mL L3000008 Neon® Transfection System 10 µL Kit 25 x 2 reactions MPK1025 Lipofectamine® 3000 Transfection Reagent 1.5 mL L3000015 Neon® Transfection System 10 µL Kit 96 x 2 reactions MPK1096 Lipofectamine® 3000 Transfection Reagent 5 x 1.5 mL L3000075 In vivo delivery Quantity Cat. No. ® Lipofectamine 3000 Transfection Reagent 15 mL L3000150 Invivofectamine 3.0 Reagent 1 mL IVF3001 ® Lipofectamine RNAiMAX Transfection Reagent 0.1 mL 13778100 Invivofectamine 3.0 Reagent 5 x 1 mL IVF3005 Lipofectamine® RNAiMAX Transfection Reagent 0.3 mL 13778030 Transfection-related products Quantity Cat. No. Lipofectamine® RNAiMAX Transfection Reagent 0.75 mL 13778075 Opti-MEM® I Reduced-Serum Medium 100 mL 31985062 Lipofectamine® RNAiMAX Transfection Reagent 1.5 mL 13778150 Blasticidin S HCl (10 mg/mL) 10 x 1 mL A1113903 Lipofectamine® RNAiMAX Transfection Reagent 15 mL 13778500 Geneticin® Selective Antibiotic (G418 Sulfate) (50 mg/mL) 100 mL 10131027 Lipofectamine® MessengerMAX™ Transfection Reagent 0.1 mL LMRNA001 Hygromycin B (50 mg/mL) 20 mL 10687010 Lipofectamine® MessengerMAX™ Transfection Reagent 0.3 mL LMRNA003 Mycophenolic Acid 500 mg 11814019 Lipofectamine® MessengerMAX™ Transfection Reagent 0.75 mL LMRNA008 Puromycin Dihydrochloride 10 x 1 mL A1113803 Lipofectamine® MessengerMAX™ Transfection Reagent 1.5 mL LMRNA015 Zeocin™ Selection Reagent 8 x 1.25 mL R25001 Lipofectamine® MessengerMAX™ Transfection Reagent 15 mL LMRNA150 ExpiFectamine™ 293 Transfection Kit 1 x 1 L culture A14524 ExpiFectamine™ 293 Transfection Kit 1 x 10 L culture A14525 ExpiFectamine™ 293 Transfection Kit 5 x 10 L culture A14526 Broad-spectrum transfection reagents Quantity Cat. No. Lipofectamine® 2000 Transfection Reagent 0.3 mL 11668030 Lipofectamine® 2000 Transfection Reagent 0.75 mL 11668027 Lipofectamine® 2000 CD Transfection Reagent 1 mL 12566014 Lipofectamine® 2000 Transfection Reagent 1.5 mL 11668019 Lipofectamine® 2000 Transfection Reagent 15 mL 11668500 Electroporation Quantity Cat. No. Neon® Transfection System 1 each MPK5000 Neon® Transfection System Starter Pack 1 pack MPK5000S Neon® Transfection System 1 each MPP100 Neon® Transfection System Pipette Station 1 each MPS100 Neon® Transfection Tubes 1 pack MPT100 Neon® Transfection System 100 µL Kit 25 x 2 reactions MPK10025 Ordering information

26 27 Find out more at lifetechnologies.com

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