TITLE: 3.3 Lentivirus Production and Transduction
AUTHORS: Evelyn J. Sauter, Lisa K. Kutsche, Simon D. Klapper, Volker Busskamp

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is part 3.3 of the "</span><a href="https://www.protocols.io/private/C65DB81838BC11EBA61A0A58A9FEAC2A/protocols" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders</span></a><span>" collection of protocols.</span></div><br/><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract:</span><span> 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.</span></div></div>

[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].
?. [3.3 Lentivirus Production and Transduction]
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 . The cells are transfected using  combined with  (see Fig. 3a, b), the viral packaging (psPAX2) plasmid, and the viral envelope (pMD2G) plasmid in a 4:2:1 ratio. In detail, mix  with  in a 1.5 ml tube.
[fresh DMEM with 10% FBS]
[polyethylenimine (PEI)]
[DNA containing the plasmid of interest]
[1 mg/ml PEI solution]
[DMEM medium w/o FBS]
?. [3.3 Lentivirus Production and Transduction]
In a separate tube, mix , , and  with .
[pMD2G]
[psPAX2]
[vector of interest]
[DMEM w/o FBS]
?. [3.3 Lentivirus Production and Transduction]
Combine PEI and DNA, mix, and incubate at  for –. Subsequently, add the transfection mix dropwise to the 293T/17 cells and place the culture dish into the incubator (see Note 18).
0 Room temperature
?. [3.3 Lentivirus Production and Transduction]
The next day, replace the medium with .
[DMEM with 10% FBS]
?. [3.3 Lentivirus Production and Transduction]
Mix  with  containing , , and , and keep at  or over the weekend.
[cell culture supernatant (48 h and 72 h harvests)]
[diluted polyethyleneglycol (PEG) solution]
[50% PEG 6000 solution]
[4 M NaCl solution]
[1× PBS w/o Ca2+ and Mg2+]
4 °C
?. [3.3 Lentivirus Production and Transduction]
After , 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 . Add  and after another  collect the supernatant, filter and pool with the first collection.
4 °C
[fresh DMEM with 10% FBS]
?. [3.3 Lentivirus Production and Transduction]
Centrifuge the tubes at  (see Note 19). After the centrifugation, a white pellet should be visible.
Centrifuge: 7000 34, 10 min, 4 10
?. [3.3 Lentivirus Production and Transduction]
Carefully decant the supernatant and resuspend the pellet in  by pipetting up and down. Vigorously vortex the tubes for – to further resuspend the pellets. Lenti-X™GoStix™can be used to confirm the successful generation of viral particles.
[1× PBS w/o Ca2+ and Mg2+]
?. [3.3 Lentivirus Production and Transduction]
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
?. [3.3 Lentivirus Production and Transduction]
In order to transfect iPSCs with the viral particles, the cells should be approximately 40 ± 10% confluent. Wash the cells once with  and add mTeSR™1 medium to the well.
[PBS w/o Ca2+ and Mg2+]
?. [3.3 Lentivirus Production and Transduction]
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  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  if used immediately or stored at .
on ice
-20 °C
?. [3.3 Lentivirus Production and Transduction]
Adjust the concentration of the pAlbumin plasmid used for normalization to , 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).
?. [3.3 Lentivirus Production and Transduction]
Prepare two master mixes of , , , and  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).
[TaqMan® Universal PCR Master Mix]
[forward primer (10 μM)]
[reverse primer (10 μM)]
[probe (10 μM)]
?. [3.3 Lentivirus Production and Transduction]
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.
?. [3.3 Lentivirus Production and Transduction]
Calculate the copy number for each sample as follows: Copy number = (quantity mean of WPRE sequence/quantity mean of Alb sequence) × 2.
?. [3.3 Lentivirus Production and Transduction]
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).
?. [3.3 Lentivirus Production and Transduction]
Add mTeSR™1 to the tube containing the virus suspension, mix gently and transfer dropwise to one or more wells (see Note 20).
?. [3.3 Lentivirus Production and Transduction]
Incubate the culture dish  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).
?. [3.3 Lentivirus Production and Transduction]
Aliquot  per well of a 96-well plate and add  (concentration between  and  in ).
[master mix]
[sample DNA]
50 ng
[DNA]
8 µl
?. [3.3 Lentivirus Production and Transduction]
Seal the plate, carefully mix by vortexing and briefly spin down.
?. [3.3 Lentivirus Production and Transduction]
Run the qPCR with the settings for FAM fluorochromes and BHQ quenchers with the following program: 1 cycle of  at  followed by 50 cycles of  at  and  at  (see Note 17).
95 °C
95 °C
60 °C