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Retrovirus Packaging Kit

$1,075.00

Item Cat No.: BCPKRV

Application: Gene Delivery

Reactivity: Cell culture

BiCell Scientific’s retrovirus packaging kit provides all the components needed to generate retrovirus, including expression vectors, helper vectors, HEK293T cell line, and BiCellFectamax transfection reagent.

Oncoretroviruses (cancer-causing retroviruses), include human T-lymphotropic virus (HTLV) causing a type of leukemia in humans, and murine leukemia viruses (MLVs) in mice, are powerful tools of gene transfer. The integration of oncoretroviral genome into host cells occurs during cell division, therefore, oncoretrovirus can only transduce dividing cells, but not cells that have entered terminal differentiation, such as neurons.

Oncoretroviruses have gag, pol and env genes, coding for viral proteins in the order: 5´-gagpolenv-3´. The 5′-end of long terminal repeat (LTR) includes four regions, which are R, U5, PBS (primer binding site), and L; the 3′-end of LTR includes three regions, which are PPT (polypurine tract), U3, and R.

BiCell Scientific’s retrovirus packaging kit provides all the components needed to generate retrovirus, including expression vectors, helper vectors, HEK293T cell line, and BiCellFectamax transfection reagent.

  • pRetro-IRES-GFP vector (1 μg) (stored at -20oC)
  • pRetro-IRES-Puro vector (1 μg) (stored at -20oC)
  • Helper vector set (combination of 2 vector set to express gag, pol and vsv-g genes) (100 μg) (stored at -20oC)
  • HEK293T cells (frozen vial of 1 x 106 cells) (stored at -80oC)
  • BiCellFectamax transfection reagent (1 ml) (stored at 4oC)

For Research Use Only. Not for use in clinical diagnostics.

Protocol is given to make 1×108 retrovirus pseudotyped with VSV-G coat protein and able to transduce target cells at titer of 1×106/ml.

  • The day before transfection, plate 1.0 x 107 HEK293T cells in 15-cm culture dish containing 20 ml of growth medium (with serum) so that they will be 70-85% confluent the next day.
  • Prepare DNA-BiCellFectamax complexes as following.
  • Dilute 10 μg of pRetro-expression vector and 30 μg of Helper vector set in 1 ml of serum-free medium. Vortex gently to mix.
  • In a separate tube, add 100 μl of BiCellFectamax into 1 ml of serum-free medium. Vortex gently to mix.
  • Combine DNA solution with BiCelFectamax solution (total volume is 2 ml). Vortex gently to mix. Incubate for 20 minutes at room temperature to allow the DNA-liposome complexes to form.
  • Add the 2 ml of DNA-BiCellFectamax complexes to the 15-cm culture dish. Mix gently by rocking the plate back and forth.
  • Incubate the cells at 37°C in a CO2 incubator for overnight.
  • The next day, remove growth medium and add 20 ml of fresh growth medium (with serum).
  • Incubate the cells at 37°C in a CO2 incubator for 48hrs to allow retrovirus secretion.
  • Two days later, harvest viral supernatant and centrifuge at 250 xg for 5min to remove cell debris.
  • Filter viral supernatant through 0.45 μm filter. Add viral supernatant to a sterile ultracentrifuge tube.
  • Centrifuge at 50,000 xg for 90min at 4oC to pellet virus.
  • Aspirate remaining medium. Add 100 μl of ice-cold PBS to the white pellet. Resuspend the pellet by pipetting up and down.
  • Freeze virus at -80oC or use fresh virus to infect target cells.

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"I am really impressed with your approach. We tried multiple times previously to create monoclonal and polyclonal antibodies to claudin-2 and MLCK1. We have had limited success generating polyclonals and no success generating monoclonals. You have generated outstanding monoclonals to both. I look forward to continuing to work with you."

Jerrold R. Turner, M.D., Ph.D.

Brigham and Women’s Hospital | Harvard Medical School

"The polyclonal antibody you generated for KIAA0408 is stunning! KIAA0408 is a novel cilium molecule that has never been studied. So, clearly there will be a lot of demand for it as we have discovered a very interesting finding and the story will be published in a high impact journal. I am strongly inclined to generate monoclonal antibody for this protein too and we should think about patenting it."

Univ.-Prof. Jay Gopalakrishnan PhD

Heinrich-Heine-Universität | Universitätsklinikum Düsseldorf

"Your ARL13B antibody works beautifully!!! We’re so happy to have a cilia-specific antibody made in rat! I can send you high resolution images to be posted on your website."

Julie Craft Van De Weghe, PhD

School of Medicine | University of Washington

"The assay is a homophilic interaction mediated cell adhesion on purified protein (in this case, immobilized purified Pcdhga9 to Pcdhga9 expressed on cell surface). Compared to control, cell adhesion is reduced in the presence of Pcdhga9 monoclonal antibody supernatants!"

Divyesh Joshi, PhD

School of Medicine | Yale University

"The rabbit hybridoma supernatants of anti-APOBEC3 project are tested positive by ELISA, and we are very happy about it! We previously tried a company, Abclone. Their Project "A" has immune response that is <10,000 titer in antiserum, which would explain why there is no positive mAb after fusion. Their project "B" didn't have any immune response in rabbit."

Harshita B Gupta, PhD.

School of Medicine | UT Health San Antonio

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