Gene Vector Core

Overview 

Gene Vector Core

Delivering genes of interest into specific target cells is an important technology for studying the functions of genes. The core is specialized in viral vector production for gene transfer in cell culture as well as in animals.

 

Services

We are currently producing:

  • Adeno-associated viral vector (AAV, serotype 1, 2, 5, 6, 8, 9, 10, PHP.eB, 7M8, DJ and DJ/8)
  • VSVG-pseudotyped lentiviral vector (LV) with second-generation or third-generation packaging systems
  • Viral stocks
  • Helper-dependent adenoviral vector (HDAd)
  • G-deleted Rabies vector

 

Viral vectors are produced and purified by basic protocols. Infectious titer of viral vectors is subject to an additional charge. Cloning assistance is provided in rare circumstances. The core has various expression cassettes and basic shuttle vectors for expression of your genes including shRNA. There is no additional charge for packaging plasmids and helper virus.

 

Instruction to Investigators


Adeno-Associated Virus (AAV)
We accept any AAV transfer/shuttle vector. For regular AAV, genome size (left ITR to right ITR) should be between 2.4 – 4.7 kb for efficient packaging. After construction of a transfer vector, prepare plasmid DNA (midi prep using any plasmid prep kit is good, though we routinely use an endo-free DNA midi kit from Omega #D6915) and precipitate by ethanol (add 1/10 volume of 3M sodium acetate buffer, pH 5.2 and 2 volume of ethanol). Endotoxin-free preparation is preferable, but not required. Rinse the DNA pellet with 70% ethanol. From this stage, work under a cell culture hood to maintain sterility. Re-suspend DNA pellets in sterile transfection grade water. We have been using iMFectin poly DNA transfection reagent (GenDEPOT) for transfection and need 50 μg total DNA at a concentration of 0.5-1 μg/μl for 20 x 15-cm dish scale (regular scale). The packaging plasmid(s) are supplied by the Core and included in the fee. We request backbone information for your shuttle vector because the titer is determined by Q-PCR. If we do not have PCR primers for your vector, please provide your vector specific Q-PCR primers (regular SYBR Green primer, not TaqMan QPCR primer). We have the following primers: EF1, CMV, CAG, hSyn, hU6, IRES, WPRE, bovine GH polyA, EGFP, and Rluc.


We split cells on Monday and Thursday for transfection. Transfection takes place on Tuesday or Friday followed by purification in the following week. Determination of physical titer takes an additional day. Total time of vector production is approximately 2 weeks from receiving DNA to delivery of purified vector with physical titer. Requests for AAV production must be made by Wednesday in order for production to begin the following Tuesday, and DNA must be received by the following Monday afternoon. For Friday transfection, please request by Friday and bring DNA by Thursday afternoon.
AAV is stable at 4oC at least for 6 months. Please freeze at -80oC for long term storage. However, freezing/thawing should be kept to a minimum.

 

Lentiviral Vector (LV)
We accept any lenti shuttle vector (also known as transfer vector). The genome size of packaged LV should be less than 7.5 kb (5’ LTR to 3’ LTR) for high titer packaging. LV is a single-stranded RNA virus. Poly A signal should not be included in your gene of interest since it inhibits elongation of the viral genome during vector production.


LV shuttle vector grows well in E. coli (DH5α). After construction of a shuttle vector, precipitate DNA by ethanol (add 1/10 volume of 3M sodium acetate buffer, pH 5.2 and 2 volumes of ethanol). Rinse the DNA pellet with 70% ethanol and then transfer the tube under a cell culture hood to maintain sterility. Re-suspend the DNA pellets in sterile transfection-grade water. We need at least 15 μg of transfection ready DNA for a 2 x15-cm dish at a concentration of 0.5-1 μg/μl (no higher than 2 μg/μl, please). The standard scale is 2 x 15-cm dishes. The packaging plasmids (psPAX2 and pMD2.G for the second generation, and tat, Gag, Pol, pMD2.G for the 4th generation) are supplied by the Core and included in the fee. However, we routinely use 2nd generation packaging to achieve the highest titer.


Transfection takes place on Tuesdays and purification/concentration on Thursdays of each week. Determination of infectious titer takes an additional week. Total time of vector production is approximately 2 weeks from receiving DNA to delivery of purified vector with titer. Requests for LV production must be made by Thursday in order for production to begin the following week, and DNA must be received by the following Monday afternoon. LV is concentrated by ultracentrifugation over a sucrose gradient, re-suspended in 0.3 mL (0.15 mL/15-cm dish) of plain DMEM or a medium of the customer’s choice, aliquoted in 3 x 100 μl and stored at -80oC.


2 x 15-cm dish-scale transfection yields approximately 0.3 mL of virus solution at a concentration of 5 x 108 – 1 x 109 transducing units/mL. The titer is influenced by lenti genome size and transgene. In our hands, lenti vectors containing repeat sequences or regulatory systems (such as rtTA or tTA) tend to have low titer.


Titration for infectious titer is not included in regular production. The end-point transduction assay by real time PCR is available for an additional charge.


Concentrated LV is stable at -80oC for at least a year. We found that freezing/thawing three times or left at 4oC for 1 day does not significantly reduce the titer but recommend to aliquot for convenient volume (but not too small to avoid pipetting loss and evaporation), and refreeze at -80oC. Freezing/thawing should be kept to no more than 3 times.

Getting Started

  • BCM Users:  Click the link in the upper right corner to login or register for an iLab account using valid BCM credentials.
  • Non-BCM Users:  Click the appropriate link in the upper right corner to login with your approved iLab credentials or to sign up for an iLab account.

 

Leadership

Dr. Kazuhiro Oka, Director
ABBR-R617
Phone:  713-798-7381
Email: kazuhiro@bcm.edu 

Location and Hours of Operation

Location

Hours

Baylor College of Medicine

Margaret M. Alkek Building for Biomedical Research (ABBR)

One Baylor Plaza

Houston, TX 77030  


Gene Vector Lab: ABBR Room R630

Phone: 713-798-4409      

9 a.m. - 5 p.m., Monday to Friday   

 

 

Links and Resources

 

Publication Acknowledgements

Scientific publications containing data obtained using the services offered by the core should include the following statement in the acknowledgment section: the ATC Gene Vector Core at Baylor College of Medicine.

 

Contacts

Name Role Phone Email Location
Kazuhiro Oka
Director
 
713-798-7381
 
kazuhiro@bcm.edu
 
ABBR-R617
 
Ashkaan Moinzadeh
Technician
 
713-798-4409
 
Ashkaan.Moinzadeh@bcm.edu
 
ABBR-R631K
 
Internal BCM iLab Support
Internal BCM iLab Support
 

 
ilabs-support@bcm.edu
 

 
Austin Seal
Research Assistant
 
281-639-2703
 
Austin.Seal@bcm.edu
 
ABBR-R631K