Services

The shRNA and ORF reagents are available to The University of Texas MD Anderson Cancer Center researchers ONLY due to licensing agreements.
If any new services are needed, please contact Dr. Yutong Sun (FGC co-director) ysun2@mdanderson.org. The FGC will  be open to discuss/provide new services upon inquiries.  

Individual clone distribution as glycerol stocks and plasmids

FGC distributes individual shRNA or ORF in forms of glycerol stock (in LB medium containing 8% glycerol)  or plasmid (midi-prep, >100ug).  ORF plasmids will be verified by partial sequencing before they are released to researchers. The turnaround time is 1-2 business days for the glycerol stock and 2-3 business days for the plasmids.

You can search for GIPZ shRNAs (human and mouse), Human ORFeome Collaboration and Human Precision LentiORF at https://horizondiscovery.com/ by gene symbols. ORFeome Collaboration ORFs are in a Gateway Entry vector and Precision LentiORF ORFs are in a lentiviral vector.

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Individual Lenti- or retro-virus packaging and transduction

FGC conducts plasmid purification, lenti- or retro-virus packaging and titer measurements. We also conduct this service if researchers provide us with their own viral vectors. The turnaround time is 4-6 weeks. The FGC will not bill users if the virus packaging is failed, i.e. functional titer is too low.

Virus particles are provided in two forms: non-concentrated virus in the condition medium (8x1ml) and concentrated virus resuspended in DMEM or the requested medium (8x100ul). Virus titer is functionally determined in 293T cells using a fluorescent marker or in Hela cells using antibiotics selection. Depending on the lenti- or retroviral vector, the virus titer varies. For example, concentrated GIPZ shRNA lentivirus titer usually ranges from 5x10^7TU/ml to 2x10^8TU/ml in 293T cells and non-concentrated TKO v3 lentivirus titer is over 5x10^6TU/ml in 293T cells. However, the virus titer could be as low as 10^5TU/ml when using other lenti- or retro-viral vectors.

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Note: If you need a custom size of the lenti- or retro-virus packaging, please contact Dr. Yutong Sun ysun2@mdanderson.org to discuss a quote.

Adeno-associated Virus (AAV) Packaging & Purification

Depending on the AAV production scale, researchers need to provide us with 150-500ug of the pAAV plasmid (endotoxin-free midi-, maxi- or mega-prep) with the vector map/sequence. If the plasmid is not enough, FGC can start AAV packaging from plasmid prep. We can also help to clone ORF/shRNA/sgRNA into AAV vectors. Quotes will be provided for additional services.

Turnaround time: The turnaround time for AAV production process will take 2-3weeks starting from AAV plasmid transfection into HEK293 cells. Additional time will be needed if vector cloning or plasmid prep is requested.

AAV Serotype: DJ; pAAV2/1; pAAV2/2; pAAV2/5; pAAV2/7; pAAV2/8; pAAV2/9n; pAAV2/rh10; pAAV2/retro helper; pDGM3B; pDGM6; pRepCap6; 7M8; shh10 and pAnc80L65AAP

^#pre-packed control AAV: pAAV CMV-NLuc-P2A-copGFP-T2A-PuroR Serotype DJ; pAAV2/8; pAAV2/9n; pRepCap6;
 

Customized Sub-libraries (glycerol stock, plasmids and/or lentivirus)

Based on the needs of researchers, FGC will “cherry-pick” shRNA or ORF glycerol stocks in a 96-well plate format. Depending on the researchers’ requests, we will purified the plasmids (transfection compatible) in 96-well plate format, and/or conduct lentiviral packaging in 96-well plates.

Price: please contact Dr. Yutong Sun ysun2@mdanderson.org to discuss a quote.

Establishing Somatic Gene-knockout Cell lines using the CRISPR/Cas9 technology

FGC will provide a clonal somatic gene-knockout cell line with validated gene-knockout by PCR (HR method) or sequencing (NHEJ method). The FGC will not charge users before delivering validated KO cell lines.

Requirements: 1) cell line can tolerate clonal selections; 2) Loss of target gene will not kill the cells or significantly decrease cell proliferation rate;  3) cell line is compatible with transfection or transduction; 4)Western blots (for proteins), qPCR (for non-coding RNAs) or other assays are available to detect the loss of a target gene.

FGC will design and subclone gRNAs into a LentiCRISPR v2 vector, determine gRNA's gene editing ability by T7 endonuclease assay, transfect or transduce cell lines, perform clonal selections by western blots or qPCR, carry out gDNA sequencing around the DSB site to confirm gene editing and authenticate the cell line(s) at the MD Anderson Cytogenetics and Cell Line Authentication Core.

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Project services

FGC staff will spend some of their efforts to facilitate MD Anderson Cancer Center researchers optimizing screen assays, perform screens and data analysis.

Other Services

Custom shRNA or ORF sub-cloning

FGC subclones custom shRNAs into the pLKO.1 vectors, EZ-TET-pLKO vectors (an inducible shRNA vector), an ORF into a pCDH vector or custom gRNAs into LentiGuide-puro or LentiCRISPR v2.

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Note: If you need to engineer a vector or to carry out a complicated/large subcloning project, please contact Dr. Yutong Sun ysun2@mdanderson.org to discuss a quote.

Shared Equipment

The following instruments are open to MD Anderson researchers: robots (Tecan EVO100 and EVO200) and High-content imaging systems (ImageXpress micro and ultra).

Pre-assembled shRNA libraries

FGC distributes the following pre-assembled shRNA libraries targeting the following gene families: Human kinases; Human phosphatases; and human E3 ligases. In addition, we also distribute Cellecta human Genome-Wide Pooled shRNA library (lentivirus).

TKO v3 CRISPR Library

FGC distributes the TKO v3 CRISPR Library (lentivirus, Dr. Moffat’s lab at the University of Toronto, https://www.addgene.org/pooled-library/moffat-crispr-knockout-tkov3/).

To request our services, please login your iLab account using your MD Anderson username and password and request our services through the MD Anderson Functional Genomics Core.