The Genome Engineering and induced pluripotent stem cell Center (GEiC) supports researchers with up-to-date technologies to meet their cell and animal model needs. On the genome engineering front, we offer a variety of services tailored to best fit investigators individual preference, ranging from CRISPR reagents with or without validation, to cancer/iPS cell line modification or animal model creation. We work closely with mouse cores from design, reagent validation to genotyping, process improvement and troubleshooting, when necessary. We also provide next-generation sequencing (NGS) based human cell authentication and NGS-based genotyping services and general molecular biology assistance. On the iPSC front, we culture, bank and store primary fibroblasts and renal epithelial cells from patient samples for investigators, and iPSC reprogramming service is provided from fibroblasts, RECs as well as peripheral blood mononuclear cells (PBMCs). The GEiC aims at helping investigators convert their idea to a useful model quickly and cost-effectively.
With this service, we introduce investigator-defined genomic modifications into investigator-specified cancer cell lines. The deliverables are up to two clones with desired modifications.
We differentiate iPSCs into cardiomyocytes. The deliverable is a 6-well plate of differentiated cardiomyocytes.
After investigators complete the screening in cultured cells, we perform bioinformatics analysis of the post screening cell pool and compare gRNA representation to the original library. The deliverable is list of gRNAs that are enriched or absent after screening, indicative of functional involvement of respective genes in the given biological process.
CRISPR screen is a powerful technology that allows one to identify a gene or a set of genes that are critical for a biological process with a distinguished phenotype in the culture. We design gRNAs against investigator-defined set of genes and clone the into an expression library. The pooled library will be analyzed by NGS to confirm representation of all designed gRNAs. The deliverable is NGS-validated library that investigators can use for viral preparation for downstream screening.
Once gRNA is chosen (from validation step), we design homologous templates (donors) carrying specific modifications for knockin projects. A donor can be a double stranded plasmid, single stranded DNA oligo (ssODN) and long single stranded DNA (lssDNA). We outsource the production of ds plasmid, ssODN and generate lssDNA from a double stranded plasmid. We then co-transfect the donor with corresponding gRNA and Cas9 into cultured cells to validate knockin events. The deliverables are validated donor and optimized genotyping protocol for detecting knockin events.
This service is especially critical for mouse projects.
Fibroblasts isolated from skin biopsy are reprogrammed into iPSCs using Sendai virus cocktail. The deliverables are at least two clones of iPSCs.
We provide molecular biology consultation and help with plasmid design, synthesis and preparation, cell line transfection, etc., to labs that are not equipped with molecular biology skills. It is a highly customized service.
Based on the customer's need, we decide the region in the target gene sequence to run our in-house gRNA design algorithm, which outputs the potential gRNAs. We then assemble two gRNAs in either expression plasmid constructs or as in vitro transcripts and transfect them with Cas9 into cultured cells to validate cleavage activity at the specific target site. The deliverables are active gRNAs in either format that cleaves desired sequences.
We provide CRISPR reagents to mouse cores to create various genetically modified mouse models. We use precomplexed in vitro transcripts of gRNAs and Cas9 protein for all mouse projects.
With this service, we introduce investigator-defined genomic modifications into investigator-specified iPSC cell lines. The deliverables are up to two clones with desired modifications.
We test every incoming cell line for mycoplasma contamination and also test every clone before delivery. This is also a stand-alone service to test the investigator-submitted cultures for contamination. The deliverable is whether or not a culture is contaminated.
We differentiate iPSCs into neural progenitor cells. The deliverable is a 6-well plate of differentiated neural progenitor cells.
We differentiate iPSCs into neural stem cells. The deliverable is a 6-well plate of differentiated neural stem cells.
We take investigators modified cell lines and mouse tail snips to PCR verify whether desired modifications are at the expected loci. Most of the tail samples are from mice created by using CRISPR reagents we validated, which we already have primers and PCR protocols. We design PCR protocols to genotype cell samples based on information provided by investigators. For most of modifications, the PCR products were analyzed by NGS, whereas some larger knockin projects, we use junction PCRs to detect specific integration. The deliverables are detailed genotype at the target sites and a conclusion whether or not knockin is detected and at what percentage.
PBMCs isolated from blood are reprogrammed into iPSCs using Sendai virus cocktail. The deliverables are at least two clones of iPSCs.
This is a QC for pluripotency of iPSCs. Four stem cell markers are immune-stained on iPSCs. A good iPSC clone should be positive for all four. The deliverables are pictures of staining and conclusion whether a given iPSC clone has all four pluripotency markers.
Renal epithelial cells isolated from urine are reprogrammed into iPSCs using Sendai virus cocktail. The deliverables are at least two clones of iPSCs.
Patient information-blinded skin biopsy samples are digested, and fibroblasts are cultured. When enough cells are obtained, they will be cryopreserved for downstream reprogramming or other use. The deliverables are frozen vials of fibroblast cells.
Each human individual has a unique set of short tandem repeats (STR), which is used in forensics. Human cell lines, even when immortalized maintain the same profiles as the individuals they derived from. We PCR amplify up to 13 different loci to determine the identity of a given human cell line by using NGS. The assay is also great to detect potential cross contamination between different cell lines. This is a stand-a-lone service for any investigator submitted samples. We also use the assay to verify the cell line identity before starting a project and before delivery of modified clones to ensure our customers receive exactly the lines they need. The deliverables are exact numbers of repeats at all loci tested and whether the cells are the expected lines and whether there is cross contamination from a different line.
Patient information-blinded urine samples are spun down, and renal epithelial cells are cultured. When enough cells are obtained, they will be cryopreserved for downstream reprogramming or other use. The deliverables are frozen vials of renal epithelial cells.
We isolate peripheral blood mononuclear cells (PBMCs) from whole blood, expand and cryopreserve the cells. The deliverables are frozen vials of PBMCs.