Feb 28, 2024

Public workspaceGenetic Modification of Human Induced Pluripotent Stem Cells (hiPSCs) by Lentiviral Transduction Protocol

DOI
dx.doi.org/10.17504/protocols.io.n92ldm428l5b/v1
  • Roshni Jaffery1,2,
  • Ningbo Zheng1,2,
  • Jiakai Hou1,2,
  • Ashley Guerrero1,2,
  • Si Chen1,2,
  • Chunyu Xu1,2,
  • Nicholas A. Egan1,2,
  • Ritu Bohat1,2,
  • Weiyi Peng1,2
  • 1Department of Biology and Biochemistry, University of Houston, Houston, TX, USA;
  • 2Aligning Science Across Parkinson's Collaborative Research Network
Sarfraz Ahmed
Open access
DOI: dx.doi.org/10.17504/protocols.io.n92ldm428l5b/v1
Protocol Citation: Roshni Jaffery, Ningbo Zheng, Jiakai Hou, Ashley Guerrero, Si Chen, Chunyu Xu, Nicholas A. Egan, Ritu Bohat, Weiyi Peng 2024. Genetic Modification of Human Induced Pluripotent Stem Cells (hiPSCs) by Lentiviral Transduction Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldm428l5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 28, 2024
Last Modified: February 28, 2024
Protocol Integer ID: 95911
Keywords: hiPSCs, lentivrial transduction, genetic modification
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000312
Abstract
This is the protocol for the genetic modification of human induced pluripotent stem cells (hiPSCs) using lentiviral transduction method.
Materials
1. Cell Lines: 293T and hiPSCs of interest
2. Plasmid of interest
3. Packaging plasmids: VSVG and psPAX2
4. JetPRIME buffer
5. jetPRIME transfection reagent
6. DMEM media + 10% FBS complete mixture
7. 0.45 um PDVF membrane filter
8. 5 mL syringe
9. MilliporeSigma‱ Amicon‱ Ultra-15 Centrifugal Filter Units
10. StemFlex media and supplement (ThermoFisher Scientific Cat.No.A3349401)
11. Vitronectin (VTN)
12. Polybrene
Day 1
Day 1
Grow and culture 293T cell line in 10 cm plates.
Grow and culture hiPSCs in pre-coated VTN 6 well plates.
When there are enough 293T cells, seed 5 million 293T cells a 10cm plates in 10mL DMEM complete media per plasmid of interest.
Grow overnight at 37 deg C and 5% CO2 until 80%-90% confluence.
Day 2: Transfection
Day 2: Transfection
Change media of wells. Make a master mix in 1.5 mL Eppendorf tubes for each plate as follows:
ABCDE
JetPRIME Buffer (Polyplus Transfection, Ref #: 114-15) Plasmid interest Packaging plasmid VSVG Packaging plasmid psPAX2 jetPRIME transfection reagent (Polyplus Transfection, Ref #: 114-15)
500 uL 500 uL 2 ug 3 ug 1:2 ratio of total DNA (ug): transfection reagent (uL) =20 uL
Vortex 10s.
Incubate 10 min RT *NEVER OVER 30 MIN*.
Add all of mix to wells dropwise and rock plate and incubate overnight 37 deg C and 5% CO2.
Day 3
Day 3
Check cells 24 hours post-transfection.
First thing in the morning, exchange the media – add 15 mL pre-warmed DMEM complete media to transfected wells. (This removes the jetPRIME transfection reagent and residue plasmid.).
Split hiPSCs into clumps in StemFlex complete medium. Seed 20% confluency or ~0.1M per well in 2 mL of 6 well plate. Note: Cell number depends on cell type.
Day 4: Virus Collection
Day 4: Virus Collection
Check cells 48 hours post-transfection. Collect virus in 50 mL tube.
Centrifuge 1500 rpm for 5 minutes.
Using a 0.45 um PDVF membrane filter and 5 mL syringe, collect virus into new 50 mL tubes. Avoid bottom of tube with cell debris
Concentrate 1:40 [~42-45mL of diluted virus makes approximately 0.8-1mL of concentrated virus]
Collect and label MilliporeSigma‱ Amicon‱ Ultra-15 Centrifugal Filter Units.
Rinse column with PBS and spin 2,000 × g for 5 min.
Add up to 10 mL of sample to the Amicon‱ Ultra filter device.

Centrifuge at 2,000 × g maximum for 10 minutes.
o Repeat adding rest of virus to Amicon‱ Ultra filter device and centrifuge again at 2,000 × g maximum for 10 minutes until all virus is concentrated.

Collect concentrated virus and aliquot 0.2mL per Eppendorf tube. Use virus, other tubes store -80degC for future use if necessary.
Day 4 (continued): Virus infection
Day 4 (continued): Virus infection
Remove the culture medium from cells, add 850uL fresh Stemflex complete medium into each well of 6 well plate.
Add 150ul virus and 1ug/ml polybrene into cells. Rock plate.
Centrifuge at 1000rcf for 90min at room temperature.
Incubate at 37 C0, 5% CO2 overnight.
Day 5: Change medium
Day 5: Change medium
Replace the medium with 2ml fresh Stemflex complete medium.
Day 6: Check cells
Day 6: Check cells
Check the expression of plasmid via microscopy (if applicable).
Begin selection (if applicable).
Day 7 and onward
Day 7 and onward
Check the cell status.
Split cells if necessary.
Freeze cells to store.