Resource Summary Report  New Search Previous Search Results

URL: https://dx.doi.org/DOI:10.17504/protocols.io.5fvg3n6

Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward

Group: Neurodegeneration Method Development Community

Summary: 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 (Basic Protocol 4).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.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 cat. no. 105842 for direct use with hNIL or excision and insertion into other constructs.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 Support Protocol 6).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.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.This protocol will continue with the assumption of integration of a transgene cassette into a safe-harbor locus as described in Basic Protocol 2. 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 Basic Protocol 4 for clonal isolation.

Affiliations: National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland

External URL: https://doi.org/10.1002/cpcb.51

Version: 1

Publication Date: 2019

Expand All Collapse