Color-Switch CRE Reporter Stable Cell Line

Color-Switch CRE Reporter Stable Cell Line Catalog Number SC018-Bsd SC018-Neo SC018-Puro Product Name / Description CRE reporter cell line (Bsd): HEK293-loxP-GFP- RFP (Bsd). Cell line expresses "LoxP-GFP-stop- LoxP-RFP" cassette with blasticidin antibiotic. CRE reporter cell line: HEK293-loxP-GFP- RFP (Neo). Cell line expresses "LoxP-GFP-stop- LoxP-RFP" cassette with Neomycin antibiotic. CRE reporter cell line: HEK293-loxP-GFP- RFP (Puro). Cell line expresses "LoxP-GFP-stop- LoxP-RFP" cassette with Puromycin antibiotic. Amount 1 vial of cells (1.0 ml, 2 x 10 6 cells) in 80% DMEM, 10% FBS, 10% DMSO 1 vial of cells (1.0 ml, 2 x 10 6 cells) in 80% DMEM, 10% FBS, 10% DMSO 1 vial of cells (1.0 ml, 2 x 10 6 cells) in 80% DMEM, 10% FBS, 10% DMSO Storage: Liquid Nitrogen; Product Description CRE recombinase, from bacteriophage P1, catalyzes recombination between 34 base-pair target sequences called lox sites and can join individual plasmids containing lox sites. CRE recombination provides an excellent tool for conditional gene targeting in transgenic animal models by linking genotypic alterations to the biological outcomes (phenotypes). By inserting a "LoxP-flanked expression target" into a host's genome, target expression can be controlled via CRE recombinase. Expression of LoxP-flanked target genes may occur prior to the addition of CRE enzyme; when CRE is applied, it deletes the LoxP flanked target segment and stops the target expression. Simultaneously, CRE-mediated recombination can activate expression of a second target downstream from the deleted segment. CRE-LoxP recombination depends upon an effective CRE enzyme delivery in vivo. GenTarget provides pre-made CRE expression lentivirial particles which are engineered with a NLS (nuclear localization signal) element, providing high LoxP-GFP-LoxP-RFP / CRE reporter stable cell line manual, Page 1 of 10

efficiency delivery of the expressed CRE protein into cell nuclear where the recombination occurs. In order to monitor and to confirm the CRE-LoxP recombination event, GenTarget also generates three CRE reporting cell lines (SC018 stable cell lines). Those stable cell lines are derived from HEK293 cell [Note: GenTarget provides the premade CRE reporting lentivirus (CAT#: LVP460) for make this reporting cell line in your desired host cell types]. The HEK293 Cell Line is a permanent line established from primary embryonic human kidney transformed with sheared human adenovirus type 5 DNA. The expressed E1A adenovirus gene allows these cells to produce very high levels of protein. The CRE reporting cell line's genome has integrated with a CRE recombination response, "Color-Switch segment" (see DNA insertion scheme below). Each cell line has also integrated with an antibiotic selection under a RSV promoter (not showed in the scheme below), thus each cell line has a specific antibiotic, blasticidin (Bsd), Puromycin (puro) or Neomycin (Neo) for selection purpose when applicable. How it works: The CRE reporting cell lines are used to monitor or confirm the efficiency of CRE recombination in vivo. It is a great method and easy tool to verify the performance of your CRE enzyme (your CRE expression plasmids, or pre-made CRE expression lentivirus, or purified CRE enzyme) in vivo conditions. It is also a control test to verify your CRE-loxP based system. LoxP-GFP-LoxP-RFP / CRE reporter stable cell line manual, Page 2 of 10

The cell line demonstrates strong GFP fluorescent signal in normal culture condition as the constitutive CMV promoter drives the GFP high expression to its stop codon. The downstream RFP ORF was not expressed because of the stop codon after the GFP ORF*. Once the CRE protein was present in nuclear, the CRE excises / deletes the DNA fragment between two loxp sites, which removes the stop codon (see the image above). As a result, the RFP ORF is then expressed under the CMV promoter, and the cell line switches to RFP fluorescent**. The RFP signal can be easily monitored via fluorescent cell sorting (for the ratio between GFP and RFP cells), or visualized under microscope, or measured the fluorescent intensity by a meter or reader with RFP filter set. Notes: *Like any mammalian pol II promoter, the CMV promoter could seek any possible ORFs, and in some cell types, it may slightly express the 2nd ORF (the RFP in this case) which is considered the basal or leaking RFP signal. ** It is not possible for CRE to be delivered into all cells; therefore, some GFP positive cells may still be detected even after the application of CRE. ***Also, because some cells may integrate multiple copies of the LoxP-GFP-LoxP- RFP cassette, and since 100% CRE recombination is not possible for all sites in every cell, there will be many cells demonstrating both GFP and RFP signals after the addition of CRE recombinase. The important observation is the dramatic increase in RFP positive cells following addition of CRE. And the RFP/GFP intensity ratio reflects the CRE recombination rate. Application protocol (for reference only): 1. Culture the cell in completed medium (see Culture procedures below): All three CRE reporting cell lines (SC018-Bsd, SC018-Neo and SC018-Puro) normally express strong GFP signal in normal culture conditions without any treatment. Seed cells into 24-well plate at appropriate cell density (like 1 ~ 2 x10 5 cells/per well), incubate 37oC, 5% CO2 for overnight; 2. CRE enzyme delivery: One the 2nd day when the cell density at 50% ~ 75% confluent, then deliver CRE protein using your favorite procedures which could be carried out as follows: a. Simply add 50ul of pre-made CRE expression lentiviral particles (see GenTarget's pre-made CRE expression lentivirial particles product page). LoxP-GFP-LoxP-RFP / CRE reporter stable cell line manual, Page 3 of 10

b. Transfect your CRE expression plasmid using a trasnfection lipids (like LF2k or Fugene). c. Add purified nuclear membrane penetrating CRE protein directly into cell culture. (Note: remember to set the negative control wells without adding CRE, and you may also set the positive control wells by adding 50ul of Gentarget's NLS-CRE expression lentiviral particles, Cat#: LVP297, LVP336, LVP339 and others). 3. CRE recombination reaction detection: The RFP signal will gradually show up and peaked at 48 hours or longer times (dependent upon CRE delivery methods) after the CRE delivery. The RFP/GFP cell population ratio or the RFP signal intensity reflects the CRE-LoxP recombination efficiency (rate). You can sort the cell by FACS machine, other meters, or visualize the RFP positive cell under fluorescent signal. Culture procedures 1. Thaw the frozen vial of cells quickly in a 37 C water bath (1~3min), decontaminate the outside of the vial with 70% ethanol. 2. Transfer the entire contents of the cryovial into a T-75 cm2 flask containing 15 ml of pre-warmed complete medium. Incubate the cells overnight in a 37 C incubator, 5% CO2. 3. The following day, replace the medium with 15 ml of pre-warmed, complete medium (Optional: add 10 μg/ml blasticidin in medium for SC018-Bsd cell, or 5 ug/ml of Puromycin for SC018-Puro cells or 25 ug/ml of Neomycin for SC018-Neo cells). 4. Incubate the cells and monitor cell density. 5. Pass cells (1:10 dilution) when the culture reaches 80-90% confluent. 6. For storage or stock up, freeze cells at a density of 3 x 10 6 cells/ml using 90% complete medium with 10% DMSO. Complete medium D-MEM (high glucose) 2mM L-glutamine 10% Fetal Bovine Serum (FBS) 0.1 mm MEM Non-Essential Amino Acids (NEAA) 1% Pen-strep Quality Control: Each vial contains greater than 2 x 10 6 cells with >95% viability before freeze. Cells are tested free of bacteria, viruses, mycoplasma. LoxP-GFP-LoxP-RFP / CRE reporter stable cell line manual, Page 4 of 10

Warranty and Terms: 1. This product is warranted to perform as described when used in accordance with this manual. GenTarget Inc MAKES NO REPRESENTATIONS AND EXTENDS NO WARRANTIES OF ANY KIND, EITHER EXPRESS OR IMPLIED. Gentarget s sole remedy for breach of warranty should be, at the option of Gentarget, to repair or replace the product if this product does not meet the stated quality standard. 2. By paid the purchase price, buyer is granted a non-transferable, non-exclusive license to use the product. This product is sold for research and development purposes only. 3. This product is limited to the laboratory that the product is delivered to. This Product is not for resale; or distribute, transfer for any purpose, including transfer of the Product as a component of any products; GenTarget will retains all rights for this Product's license and other intellectual property. 4. This Product should used for non-profit purpose, including any products and services usage; Furthermore, research use only means that this product is excluded, without limitation, from resale, repackaging, or modification to be used for the making or selling of any commercial product s or services without the written approval of GenTarget. You may contact our Business Development department at support@gentarget.com for product proprietary information. 5. GenTarget is not liable, and do not have any responsibility or liability, whatsoever for any direct and indirect, consequential, or other damages resulted from using this Product. Sample images of CRE-loxP recombination detection: Left panel / without CRE: CRE reporter cell line (Cat#: SC018-Bsd) was cultured in completed. Images were taken under microscope with GFP filter set (Ex 490nm/Em 525nm) and RFP filter set (Ex 545nm/Em 620nm). Right panel / with CRE: CRE reporter cell line (Cat#: SC018-Bsd) was cultured in completed in 24-well plate. 50ul of CRE expression lentiviral particle (Cat#: LVP339) was added into the cells in one well. Images were taken at ~ 72 hours after the addition of CRE expression lentivirus. References: 1. Sauer, B. (1987) "Functional expression of the Cre-Lox site-specific recombination system in the yeast Saccharomyces cerevisiae", Mol Cell Biol 7: 2087-2096 2. Stanislaw J. Kaczmarczyk and Jeffrey E. Green. Nucleic Acids Res. 2001 June 15; 29(12): e56. LoxP-GFP-LoxP-RFP / CRE reporter stable cell line manual, Page 5 of 10

Attachment: GenTarget's pre-made stable cell line list Catalog # Product Name SC001 SC002-Bsd SC002-GB SC002-GP SC002-Neo SC002-Puro SC002-RB SC002-RP SC003 SC004-Bsd SC004-GP SC004-Neo SC004-Puro SC004-RB SC004-RP SC005-Bsd SC005-GB SC005-Hygro SC005-Neo SC005-Puro SC005-RB HEK293-GFP stable cells luciferase (firefly), HEK293 stable cells (Blasticidin) luciferase (firefly), HEK293 stable cells (GFP-Blasticidin) luciferase (firefly), HEK293 stable cells (GFP-Puromycin) luciferase (firefly), HEK293 stable cells (Neomycin) luciferase (firefly), HEK293 stable cells (Puromycin) luciferase (firefly), HEK293 stable cells (RFP, Blasticidin) luciferase (firefly), HEK293 stable cells (RFP-Puromycin) LacZ stable cells CRE stable cells with Puromycin CRE stable cells with GFP-Puromycin dual CRE stable cells with Puromycin CRE stable cells with Puromycin CRE stable cells with RFP-blasticidin dual CRE stable cells with RFP-Puromycin dual HEK293-TetR (Bsd) HEK293-TetR (GFP-Bsd) HEK293-TetR (Hygro) HEK293-TetR (Neo) HEK293-TetR (Puro) HEK293-TetR (RFP-Bsd) LoxP-GFP-LoxP-RFP / CRE reporter stable cell line manual, Page 6 of 10

SC005-RP SC006 SC007 SC008 SC009 SC010 SC011 SC012 SC013 SC014 SC015 SC016 SC018-Bsd SC018-Neo SC018-Puro SC020-Puro HEK293-TetR (RFP-Puro) Flp recombinase expression HEK293 stable cell HEK293-RFP stable cells GFP-LacZ & RFP stable cells GFP & RFP HEK293 stable cells HEK293-CFP stable cells HEK293-YFP stable cells TAT stable cells Glutamine Synthesis stable cells Inducible h P53 stable cells h OCT3/4 stable cells h LIN28 stable cells Color Switch, CRE report cell line: HEK293-loxP-GFP-RFP (Bsd) Color Switch, CRE report cell line: HEK293-loxP-GFP-RFP Neo) Color Switch, CRE report cell line: HEK293-loxP-GFP-RFP (Puro) luciferase (Renilla), HEK293 stable cells (Puromycin) SC020-RP SC021-GB SC021-Puro SC021-RP luciferase (Renilla), HEK293 stable cells (RFP-Puromycin) Luciferase (firefly) and CRE co-expression stable cell line (GFP-Blasticidin) Luciferase (firefly) and CRE co-expression stable cell line (puromycin) Luciferase (firefly) and CRE co-expression stable cell line (RFP-puromycin) LoxP-GFP-LoxP-RFP / CRE reporter stable cell line manual, Page 7 of 10

SC022-RB SC023-RB SC024-RB SC025-RB SC026-RB SC027-RB SC028 HEK293-CFTR cell line with RFP and Blasticidin dual HEK293-CLCN2 cell line with RFP and Blasticidin dual HEK293-TRPC3 cell line with RFP and Puromycin dual HEK293-KCNN4 cell line with RFP and Puromycin dual HEK293-ATP2B2 cell line with RFP and Puromycin dual HEK293-TRPV1 cell line with RFP and Puromycin dual Inducible RFP HEK293 stable cell line SC029 inducible RFP HEK293 stable cell line with GFP SC030 SC031-Puro SC032-Bsd inducible GFP HEK293 stable cell line with RFP Hela-RFP stable cells Luciferase (firefly), Hela stable cells (Blasticidin) SC032-GB Luciferase-2A-GFP, Hela stable cells (Blasticidin) SC032-GN Luciferase-2A-GFP, Hela stable cells (Neomycin) SC032-GP Luciferase-2A-GFP, Hela stable cells (Puromycin) LoxP-GFP-LoxP-RFP / CRE reporter stable cell line manual, Page 8 of 10

SC032-Puro Luciferase (firefly), Hela stable cells (Puromycin) SC032-RB Luciferase-2A-RFP, Hela stable cells (Blasticidin) SC032-RN Luciferase-2A-RFP, Hela stable cells (Neomycin) SC032-RP Luciferase-2A-RFP, Hela stable cells (Puromycin) SC033 SC034-Bsd SC034-Puro SC035-Puro SC036 SC037 SC038-GB SC038-GP SC038-RB SC039-Bsd SC039-Puro SC040-Bsd SC040-Puro SC040-TetR SC041 SC042 Inducible GFP HEK293 stable cell line Hela-GFP stable cells (Blasticidin) Hela-GFP stable cells (Puromycin) Hela-TetR (Puro) stable cells Inducible GFP Hela stable cell line Inducible RFP Hela stable cell line Hela-rtTA (GFP-Bsd) stable cells Hela-rtTA (GFP-Puro) stable cells Hela-rtTA (RFP-Bsd) stable cells CHO-GFP stable cells (Blasticidin) CHO-GFP stable cells (Puromycin) MDA-MB-231 / GFP (Bsd) Stable Cell Line MDA-MB-231 / GFP (Puro) Stable Cell Line MDA-MB-231 / TetR (Puro) stable cells MDA-MB-231 / Luciferase-2A-RFP Stable Cell Line SH-SY5Y / GFP (Puromycin) stable cell line LoxP-GFP-LoxP-RFP / CRE reporter stable cell line manual, Page 9 of 10

SC043-Bsd SC043-Cas9- GP SC043-Cas9- Puro SC043-Cas9- RP SC043-LG SC043-Luc SC043-TetR SC044 SC045-Cas9- Bsd SC046 SC047-GB SC047-TetR SC048 SC049-1 SC049-2 SC049-3 SC049-4 SC049-5 TLV-C A549 / GFP stable cells (Blasticidin) A549 / Cas9 (GFP-Puro) Stable Cell Line A549 / Cas9 (Puro) Stable Cell Line A549 / Cas9 (RFP-Puro) Stable Cell Line A549 / Luciferase-2A-GFP (Puromycin) stable cell line A549 / Luciferase (Puromycin) stable cell line A549 / TetR (Puro) stable cells MDA-MB-231 / Luciferase-2A-GFP Stable Cell Line Hela / Cas9 (Bad) Stable Cell Line SH-SY5Y / RFP (Puromucin) stable cell line RKO / GFP Stable Cell Line (Blasticidin) RKO / TetR (Bsd) Stable Cell Line Jurkat T Cell /Firefly Luciferase (Puro) Stable Cell line Jurkat T / GFP Stable Cell (EF1a Promoter) Jurkat T / GFP Stable Cell (Flt1 Promoter) Jurkat T / GFP Stable Cell (CD34 Promoter) Jurkat T / GFP Stable Cell (CD68 Promoter) Jurkat T / GFP Stable Cell (Survivin Promoter) HEK293-TLV lentivirus packing cells LoxP-GFP-LoxP-RFP / CRE reporter stable cell line manual, Page 10 of 10