3.3 Lentivirus Production and Transduction
Evelyn J. Sauter, Lisa K. Kutsche, Simon D. Klapper, Volker Busskamp
Human induced pluripotent stem cells
Nucleofection
PiggyBac transposon
Lentiviral transduction
CRISPR/Cas9
Transcription factor-mediated neuronal differentiation
Astrocyte coculture
Abstract
This is part 3.3 of the "Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders" collection of protocols.
Collection Abstract: Patient-derived or genomically modified human induced pluripotent stem cells (iPSCs) offer the opportunity to study neurodevelopmental and neurodegenerative disorders. Overexpression of certain neurogenic transcription factors (TFs) in iPSCs can induce efficient differentiation into homogeneous populations of the disease-relevant neuronal cell types. Here we provide protocols for genomic manipulations of iPSCs by CRISPR/Cas9. We also introduce two methods, based on lentiviral delivery and the piggyBac transposon system, to stably integrate neurogenic TFs into human iPSCs. Furthermore, we describe the TF-mediated neuronal differentiation and maturation in combination with astrocyte cocultures.
Before start
NB Introduction, Notes, and References are in the Collection Guidelines tab
Attachments
Steps
3.3 Lentivirus Production and Transduction
For the production and transduction of lentiviruses, titration and copy number determination, we follow the protocol from the Trono lab [8].
One day prior to transfection, seed 8,000,000 293T/17 cells in a 10 cm culture dish. The next day, replace the culture medium with 4mL. The cells are transfected using 45µg combined with 15µg ( see Fig. 3a, b), the viral packaging (psPAX2) plasmid, and the viral envelope (pMD2G) plasmid in a 4:2:1 ratio. In detail, mix 45µL with 955µL in a 1.5 ml tube.
![Fig. 3Transduction of iPSCs with lentiviral vectors. (a) and (b) Schematic representation of the lentiviral constructs. (a) The pLV system consists of two constructs: pLV_hEF1a_rtTA3 (top) expresses the rtTA transactivator under the control of the constitutively active EF1α promoter and pLV_TRET_Ngn2-2A-Ngn1 (bottom) expresses the transgenes under the control of the doxycycline-inducible TRE promotor [4]. The pLV plasmids depicted to here do not contain any selection markers. If selection is required, antibiotic resistance genes should be cloned into the plasmids. (b) The pLIX403 system is an “all-in-one” doxycycline-inducible system. It expresses a puromycin resistance gene together with the rtTA transactivator under the control of the constitutively active PGK promotor and the transgene under the control of the doxycycline-inducible TRE promotor. 5′ and 3′ LTR—long terminal repeats, WPRE—woodchuck hepatitis virus posttranscriptional regulatory element. (c) Schematic representation of the serial dilution of the pAlbumin plasmid for titration and copy number determination (modified from [8])](https://static.yanyin.tech/literature_test/protocol_io_true/protocols.io.bqhdmt26/Fragile%20X%20Fig%203.png "Fig. 3Transduction of iPSCs with lentiviral vectors. (a) and (b) Schematic representation of the lentiviral constructs. (a) The pLV system consists of two constructs: pLV_hEF1a_rtTA3 (top) expresses the rtTA transactivator under the control of the constitutively active EF1α promoter and pLV_TRET_Ngn2-2A-Ngn1 (bottom) expresses the transgenes under the control of the doxycycline-inducible TRE promotor [4]. The pLV plasmids depicted to here do not contain any selection markers. If selection is required, antibiotic resistance genes should be cloned into the plasmids. (b) The pLIX403 system is an “all-in-one” doxycycline-inducible system. It expresses a puromycin resistance gene together with the rtTA transactivator under the control of the constitutively active PGK promotor and the transgene under the control of the doxycycline-inducible TRE promotor. 5′ and 3′ LTR—long terminal repeats, WPRE—woodchuck hepatitis virus posttranscriptional regulatory element. (c) Schematic representation of the serial dilution of the pAlbumin plasmid for titration and copy number determination (modified from [8])")
In a separate tube, mix 2.1µg, 4.2µg, and 8.4µg with 1mL.
Combine PEI and DNA, mix, and incubate at Room temperature for 0h 15m 0s–0h 30m 0s. Subsequently, add the transfection mix dropwise to the 293T/17 cells and place the culture dish into the incubator ( see Note 18 ).
The next day, replace the medium with 7mL.
After 24h 0m 0s, collect the supernatant, with the help of a syringe pass it through a 0.45 μm PES filter into a 50 ml Falcon tube and store at 4°C. Add 7mL and after another 24h 0m 0s collect the supernatant, filter and pool with the first collection.
Mix 14mL with 6.6mL containing 3.5mL, 1.5mL, and 1.6mL, and keep at 4°C 0h 30m 0s or over the weekend.
Centrifuge the tubes at 7000x g,4°C ( see Note 19 ). After the centrifugation, a white pellet should be visible.
Carefully decant the supernatant and resuspend the pellet in 150µL by pipetting up and down. Vigorously vortex the tubes for 0h 0m 20s–0h 0m 30s to further resuspend the pellets. Lenti-X™GoStix™can be used to confirm the successful generation of viral particles.
Transfer the virus suspension into 1.5 ml screw-cap tubes in aliquots of 50 μl, snap-freeze in crushed dry ice and store at -80°C.
In order to transfect iPSCs with the viral particles, the cells should be approximately 40 ± 10% confluent. Wash the cells once with 1x and add mTeSR™1 medium to the well.
Add mTeSR™1 to the tube containing the virus suspension, mix gently and transfer dropwise to one or more wells ( see Note 20 ).
Incubate the culture dish 0h 0m 30s and change the medium on the next day ( see Note 21 ). Starting 48 h after transduction, select for cells with an integrated construct with the appropriate antibiotic ( see Note 14 ).
For titration and copy number determination, we perform a qPCR on genomic DNA to count the number of integrated viral particles (WPRE) relative to the genome (albumin gene). Wait at least 96h 0m 0s before isolating genomic DNA using a genomicDNAextraction kit, such as the DNeasy® Blood and Tissue Kit (Qiagen), according to the manufacturer’s instructions. The DNA should be placed On ice if used immediately or stored at -20°C.
Adjust the concentration of the pAlbumin plasmid used for normalization to 1mg/mL, which corresponds to 1.2 × 1011 molecules/μl ( see Note 22 ). Prepare a standard curve, with the first point being 1 × 107 molecules in 8 μl (which corresponds to 1.25 × 106 molecules/μl). Prepare the other points of the standard curve by serial tenfold dilutions until there are ten molecules in 8 μl ( see Fig. 3c).
Prepare two master mixes of 8.5µL, 0.17µL, 0.17µL, and 0.17µL per sample including all samples and standards in duplicates (one master mix with albumin primers and probe for genomic DNA detection and one with WPRE primers and probe for lentivirus detection).
Aliquot 9µL per well of a 96-well plate and add 8µL (concentration between 50ng and 100ng in 8µL).
Seal the plate, carefully mix by vortexing and briefly spin down.
Run the qPCR with the settings for FAM fluorochromes and BHQ quenchers with the following program: 1 cycle of 0h 10m 0s at 95°C followed by 50 cycles of 0h 0m 15s at 95°C and 0h 1m 0s at 60°C ( see Note 17 ).
Plot the standard curve using the software of your qPCR machine or manually using other software such as Microsoft Excel, and calculate the quantity of albumin and WPRE for each sample using the equation of the standard curve.
Calculate the copy number for each sample as follows: Copy number = (quantity mean of WPRE sequence/quantity mean of Alb sequence) × 2.
Calculate the viral titer with the following formula: Titer (viral genome/ml) = (number of target cells counted at day 1 × number of copies per cell of the sample)/volume of supernatant (ml).
