TITLE: SELECTION AND ENRICHMENT OF TRANSGENIC CELL POPULATIONS (Basic Protocol 3)
AUTHORS: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>While lipid-mediated transient transfection and expression of transgenes in hiPSCs is often quite high (> 50 %), even an optimal transfection with the best editing tools can result in very low genomic integration efficiency (< 1 %). For this reason, establishing pure cell populations is typically the major bottleneck of any transgenic workflow. Commonly, frontline selection and enrichment are used to facilitate later single-cell cloning, which remains necessary to ensure that transgene insertion did not result in mosaicism or off-target mutagenesis (See </span><a href="https://www.protocols.io/view/basic-protocol-4-isolation-and-validation-of-clona-5f7g3rn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Basic Protocol 4</span></a><span>).</span></div><div class = "text-block">In order to remove non-edited cells, positive selection markers such as genes coding for fluorescent proteins or antibiotic resistance are often included in the insert construct under constitutive promoters, and this protocol describes general methods for the former strategy. However, constant expression of a fluorophore interferes with immunocytochemistry, and antibiotic selection can incorrectly select for cells with multiple aberrant transgene insertions. To counter these shortcomings, we developed a platform for enrichment from heterogeneous cell populations through cell-surface affinity for magnetic streptavidin beads mediated by expression of a streptavidin binding peptide (SBP) fused to the truncated extracellular and transmembrane domains of low-affinity nerve growth factor receptor (LNGFR). The SBP tag is commonly used to facilitate co-immunoprecipitation with streptavidin-coated beads, and, with fusion to the LNGFR, it is efficiently localized to the extracellular surface where it can bind the beads more readily.</div><div class = "text-block"><span>This construct was developed based on one initially designed for purification of transgenic T cells in suspension (Matheson, Peden, & Lehner, 2014), and it is included in Addgene </span><a href="https://www.addgene.org/105842/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">cat. no. 105842</span></a><span> for direct use with hNIL or excision and insertion into other constructs.</span></div><div class = "text-block"><span>Here, we extend the original protocol in order to optimize enrichment following safe harbor locus insertion in iPSCs, and we present evidence to support its use as either a positive or negative selection marker in a variety of transgenic applications. However, we have found that the SBP-LNGFR construct should be the only gene expressed under a dedicated, highly active promoter, as it appears to interfere with 2A-mediated ribosome skipping. For example, a different construct, with the reverse tetracycline transactivator (rtTA) linked to SBP-LNGFR by a T2A sequence, led to disrupted differentiation due to reduced rtTA activity (see </span><a href="https://www.protocols.io/view/support-protocol-6-assessing-rtta-activity-5w8g7hw" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Support Protocol 6</span></a><span>).</span></div><div class = "text-block">When structured correctly, however, SBP-LNGFR permits highly selective enrichment that does not require specialized equipment as in fluorescence-activated cell sorting (FACS) and does not require extensive tuning as in antibiotic selection. Notably, the efficiency of enrichment increases exponentially with a lower frequency of positive cells in the population. In addition, the protocol is easily modified to permit negative selection, e.g., for removing a floxed transgene with Cre recombinase or a gene knockout reporter. Furthermore, cells expressing SBP-LNGFR may be transiently labeled at the cell surface by incubating with any fluorophore conjugated to streptavidin. After washing to remove unbound fluorophore, these cells may be imaged, marked for clonal picking, or FACS-purified at an equivalent efficacy to cells constitutively expressing a fluorophore marker of insertion. Fluorescence then returns to undetectable levels within one passage and does not interfere with downstream immunocytochemistry applications.</div><div class = "text-block">Precise, label-free editing of endogenous loci may be enriched by FACS sorting for transient expression of the components necessary for editing 1 day after transfection [i.e., using a combined gRNA, Cas9, and GFP expression plasmid with an integration plasmid expressing RFP outside of the insertion sequence or by using functionalized RNPs such as S1mplex with Qdots or fluorescent streptavidin (Carlson-Stevermer et al., 2017)]. This can considerably increase the prevalence of correctly edited cells, but the combined stress of transfection and sorting can decrease cell survival. To compensate, it is recommended to transfect and sort (2.4 × 106 cells) in triplicate and to plate cells at high density following the sort. In addition, extensive downstream cloning and genotyping is often required due to the propensity for non-homologous end joining leading to mutagenesis at the target locus rather than homology-directed repair-mediated integration of the desired insert.</div><div class = "text-block"><span>This protocol will continue with the assumption of integration of a transgene cassette into a safe-harbor locus as described in </span><a href="https://www.protocols.io/view/basic-protocol-2-lipid-mediated-transfection-of-ip-5deg23e" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Basic Protocol 2</span></a><span>. Label-free gene knockouts caused by NHEJ-mediated indel formation are typically prevalent (> 15 %), so frontline enrichment is optional for these applications. Following enrichment, small stocks of heterogeneous cell populations should be frozen, and the cells should be taken directly to </span><a href="https://www.protocols.io/view/basic-protocol-4-isolation-and-validation-of-clona-5f7g3rn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Basic Protocol 4</span></a><span> for clonal isolation.</span></div></div>

[STEPS]
?. [Cell Preparation]
Culture cells after transfection and expand to at least one full 6-well plate or one 10 cm dish by EDTA split (see Basic Protocol 1).
Post-transfection recovery and expansion should take about 1 week; this enables the degradation / dilution of transient plasmids and permits expansion of edited cells to ensure enough cells are present for effective selection. Genomic DNA may be collected from unpurified cells to test for integration efficiency, and extra heterogeneous cells should be frozen throughout the purification process to provide a low-passage backup should contamination occur or should validation fail at any point.
?. [Cell Sorting]
When cells reach 70 % to 80 % confluency (day 3 to 4 after plating), dissociate with Accutase (see Basic Protocol 1).
?. [Cell Sorting]
Transfer cells and Accutase solution to conical tube, rinse with DMEM/F12, and spin  at , .
0 Room temperature
?. [Cell Sorting]
Aspirate supernatant and resuspend in E8 supplemented with  Y-27632 ROCK inhibitor.
Using medium at this step helps to improve cell viability; however, if FACS is not performed rapidly, cells can clump and reduce purification efficacy. Cold PBS with 3 % BSA can be substituted to reduce clumping.
?. [Cell Sorting]
Transfer cell solution to FACS tube and place on ice.
?. [Cell Sorting]
Perform FACS using appropriate fluorophore gating and nozzle diameter (see Critical Parameters and Troubleshooting for more detail).
Sort directly onto a Matrigel-coated cell culture plate loaded with E8 supplemented with   Y-27632 ROCK inhibitor (even if PBS is used in step 4 instead of E8) sincetypically small volumes are deposited. Alternatively, positive cells can be deposited into a separate tube, centrifuged, and replated. Some iPSC lines tolerate single cell/well sorting into 96- or 384-well dishes containing E8 supplemented with   Y-27632 ROCK inhibitor, which can further expedite clonal isolation and expansion. Survival of a polyclonal population can then be maximized by depositing any additional positive cells into the remaining well.
?. [Cell Sorting]
Expand selected cells for clonal isolation.
Colonies of positive cells may be marked and picked to keep, and/or negative cells maybe marked and removed.
?. [Cell Sorting]
Prepare bead incubation buffer:
?. [Cell Sorting]
Prepare bead incubation buffer by supplementing PBS with  EDTA and 0.5 % BSA.
For example, 47.4 ml PBS, 100 μl of 1M EDTA stock, and 2.5 ml of 10 % BSA stock.Buffer may be formulated by the consumer or purchased (e.g., autoMACS buffer).
?. [Cell Sorting]
Vacuum filter.
Components may already be sterile, but applying vacuum helps to degas the solution and reduce bubbling during subsequent use.
?. [Cell Sorting]
Store at .
4 °C
?. [Cell Sorting]
Bead preparation:
?. [Cell Sorting]
Resuspend the bead stock by vortexing or inversion.
Several streptavidin-coated magnetic beads are commercially available, but in our experience Dynabeads MyOne Streptavidin C1 has provided the best cell binding and enrichment efficiency.
?. [Cell Sorting]
Pipet the total amount of bead solution to be used into a sterile microcentrifuge tube.
beads per sample is recommended for positive selection, and   beads per sample is recommended for negative selection. In general, efficacy is maximized by using fewer beads for positive selection and more beads for negative selection; however, this should be balanced with reduced recovery and survival for small volumes and increased reagent cost for large volumes.
?. [Cell Sorting]
Add  bead incubation buffer to beads and invert several times to mix.
1 ml
?. [Cell Sorting]
Place the tube on a magnetic rack, invert two to three times, and let rest for at least .
?. [Cell Sorting]
Aspirate and discard supernatant.
?. [Cell Sorting]
Remove tube from magnetic rack.
?. [Cell Sorting]
Repeat steps 3.3 to 3.6.
For bead volumes above 100 μl, repeat at least twice.
?. [Cell Sorting]
Resuspend beads in buffer equal to the initial volume of beads.
?. [Cell Sorting]
Cell preparation and binding:
?. [Cell Sorting]
Prepare cells as for an EDTA split (see Basic Protocol 1, steps 15 and 16) and incubate at  for  to , until colonies begin to detach without physical dissociation.
37 °C
Do not use Accutase or other enzymatic dissociation reagents, as extracellular SBP-LNGFR is easily digested.
?. [Cell Sorting]
Prepare beads during incubation (see above).
?. [Cell Sorting]
Tilt the plate and pipet the EDTA solution two to three times down the well to dissociate and collect cells. Transfer to 15-ml conical tube.
Cells may be optionally filtered through a cell strainer to remove remaining clumps, butproper incubation and trituration should result in full homogenization.
?. [Cell Sorting]
Wash with PBS to collect remaining cells, and add to the conical tube.
?. [Cell Sorting]
Count cells (Phelan & May, 2015) and calculate total number of cells.
Initially, 5 × 106 cells per sample is recommended for both positive and negativeselection. Efficacy may be maximized by using higher cell numbers for positive selectionand by using fewer cells for negative selection; however, at least 1 × 106 cells shouldbe used to improve recovery.
?. [Cell Sorting]
Centrifuge  at , , and discard supernatant.
0 Room temperature
?. [Cell Sorting]
Resuspend with  buffer per sample and transfer each sample to a separate sterile 1.5-ml microcentrifuge tube.
1 ml
?. [Cell Sorting]
Add washed beads (see above for volume considerations and preparation instructions).
?. [Cell Sorting]
Incubate at  for  on a benchtop spinner at approximately .
0 Room temperature
Efficacy may be maximized by reducing incubation time to 10 min for positive selectionand by increasing time to 45 min for negative selection. Shorter times may not permitadequate binding, and longer times may lead to reduced survival due to osmotic stress.
?. [Cell Sorting]
Place on magnet, invert two to three times, and let sit for at least .
Beads should be completely removed from suspension and stuck against the side of the microcentrifuge tube. Buffer should be clear or white but not golden or brown.
?. [Cell Sorting]
Positive selection:
?. [Cell Sorting]
Aspirate negative cell fraction.
Suction may be used to increase throughput, but use narrow pipet tips to avoid interfering with the beads.
?. [Cell Sorting]
Add , remove from magnetic rack, and invert several times to fully resuspend beads.
1 ml
?. [Cell Sorting]
Repeat steps 5.1 and 5.2 at least three times.
The optimal number of washes depends on the initial number of cells used, with three washes ideal for 5 × 106 cells. In general, repeat washes until the removed negative fraction is clear. Performing four or more washes may improve enrichment if 1 × 107 or more cells were used, but it also reduces recovery.
?. [Cell Sorting]
Remove tube from magnetic rack, and either perform an optional additional incubation to release cells from beads (steps 5.5 to 5.8) or resuspend in E8 and plate directly (skip to step 5.9).
Directly plating cells with beads has not been shown to interfere with cell viability, and cells can be grown and passaged normally. However, removal of beads may be desired for downstream analysis applications or to hasten cell growth.
?. [Cell Sorting]
Resuspend remaining beads and cells with E8 supplemented with  Y-27632 ROCK inhibitor and  biotin.
Biotin is able to outcompete SBP for streptavidin binding sites on the beads, resulting in cell release.
?. [Cell Sorting]
Incubate at  for  on a benchtop spinner at approximately .
0 Room temperature
?. [Cell Sorting]
Place on magnet for , invert two to three times, and let sit for 1 additionalmin.
?. [Cell Sorting]
Remove supernatant with P1000.
?. [Cell Sorting]
Pipet cells and medium to a recipient culture well.
Biotin is normally present in culture medium at low levels, so supplementation should not affect the cells. If the high levels are of concern, the cells may be centrifuged and plated in fresh E8 supplemented with   Y-27632 ROCK inhibitor.
?. [Cell Sorting]
Return recipient plate to  incubator and evenly distribute cells by gently shaking plate front-to-back and side-to-side.
37 °C
?. [Cell Sorting]
The following day, wash twice with PBS, then add fresh E8 supplemented with  Y-27632 ROCK inhibitor. Repeat this step daily until colonies are well established, then return to normal E8 culture conditions.
?. [Cell Sorting]
Negative selection:
?. [Cell Sorting]
Pipet supernatant (negative fraction) into a 15-ml conical tube.
Pipet from the top of the tube and leave   to   at the bottom of the tube to reducecarryover of beads. Normally, fewer than 0.1 % of beads are carried over into the first negative fraction. No further washes are necessary, and the positive fraction may be discarded.
?. [Cell Sorting]
Add  E8 to the tube to improve cell pelleting.
1 ml
?. [Cell Sorting]
Centrifuge tube  at , .
0 Room temperature
?. [Cell Sorting]
Aspirate buffer and resuspend in E8 supplemented with  Y-27632 ROCK inhibitor.
?. [Cell Sorting]
Pipet cells and medium to a recipient culture well.
?. [Cell Sorting]
Return recipient plate to  incubator and evenly distribute cells by gently shaking plate front-to-back and side-to-side.
37 °C