Human Primary Cells
Primary cells are isolated from tissues, including blood and bone marrow. The isolated human primary cells have the capacity to function similarly to cells in vivo under the right conditions, and they can be incorporated into various areas of research as model systems. Primary cells can be used for drug discovery, toxicity testing, gene therapy, cancer research, and more.
In order to address the needs of the research community, Applied StemCell (ASC) now offers high-quality fresh and frozen human primary cells, including peripheral blood cells, cord blood cells, and bone marrow cells. ASC also provides hematopoietic stem cells, progenitor cells, and immune cells. For fresh cells, we offer on-demand, customized sample collection and processing services. All donors are IRB-approved and have been tested for Syphilis, Hepatitis B (HBV), Hepatitis C (HCV), HTLV-I/II, HIV-1, and HIV-2. Same-day delivery is available for Bay Area customers. Overnight delivery and international shipping are available for all other customers. Contact us today to learn more.
- Fresh & Frozen Primary Cells
- Custom Sample Collection & On-site Processing
- Bay Area Same-day Delivery, Call 1-866-497-4180
Workflow for iPSC Knock-in or Point Mutation
Products and Services
| Catalog ID# | Product Name | Price | |
|---|---|---|---|
|
|
|||
|
|
|||
Case Studies
Case Study #1: GFP Reporter Knock-in in iPSCs
Goal: The purpose of this project was to genetically introduce a GFP reporter tag into control human iPSCs at a specified locus "A".
Two gRNA candidates were selected based on the proximity to the knock-in sites and off-target profiles, and functionally validated in a model cell line. The gRNAs that produced the desired NHEJ frequencies were then used for the transfection into the control human iPSC line. Single cell colonies were screened by genotyping
Figure 1. PCR genotyping screening of GFP knock-in at locus "A". Three sets of PCR primers for each knock-in line were designed to amplify PCR fragments flanking left homology region (5 arm), right homology region (3 arm), and reporter gene insertion region (M) (to identify homozygous clones). Both Clone#1 and 2 were homozygous as attested by absence of PCR band (when compared to WT) and further by Sanger Sequencing (not shown).Case Study #2: Point Mutation Correction of a Mutant Allele in a Human Induced Pluripotent Stem Cell Line
Goal: The goal of this project was to correct a point mutation (single nucleotide polymorphism; SNP) found in a mutant allele of the gene-of-interest in a patient-derived iPSC line.
The point mutation was corrected by co-transfection of CRISPR reagents: Cas9, validated gRNA, and a single stranded oligodeoxynucleotide donor (ssODN) into the iPSCs. The gRNAs were designed based on the proximity to the mutation seen in the mutant allele and functionally validated for optimal NHEJ frequency. The ssODN was designed to replace the mutation with wildtype sequence using homology directed repair (HDR). Off-target analysis was also performed for each gRNA (not shown). After transfection, single clones were isolated and genotyped to confirm desired mutation correction. One corrected clone was identified and confirmed by sequencing (Figure 2A and 2B).
A.
B.
Figure 2. (A) Sequencing chromatogram of corrected clone (CTG > CCG). Red arrow indicates the nucleotide correction from a CTG to CCG. (B) Sequence alignment of corrected clone (bottom) against the parental SNP sequence (top). Two silent mutations were introduced by donor ssODN (CGC > CGT, GGG to GAG).
Case Study #3: Point Mutation Correction in a Patient-Derived iPSC
Goal: To correct a point mutation (AGC > GGC) in a gene associated with neurological disorders in a patient derived iPSC line and generate a homozygous wildtype line.
1. Patient-derived iPSCs were obtained from the patient and the cell line validated. The sequence at the desired locus (neurological gene) was confirmed to be a heterozygous mutation (AGC/GGC).
Figure 1. Schematic representation of the workflow undertaken for the project.
2. After cell line validation and confirming presence of mutation in the desired gene, two gRNAs were designed and validated by mismatch detection assay in K562 cells and frequency of NHEJ events as a result of the gRNA-Cas9 complex and DSB was quantified using next generation/ deep sequencing (NGS).
Figure 2. gRNA Activity via NGS, Normalized by NHEJ frequency resulting from control (GFP) transfection. Results showed that NHEJ frequency mediated by gRNA g1 and g2 were 27% and 33%, respectively. The gRNA g2 was selected for transfection.
3. The Cas/gRNA and donor vectors were transfected into the hiPSCs. After a transient puromycin selection, single cell colonies were isolated and expanded. Individual colonies were then genotyped by PCR and sequencing. Positive clones were expanded, and the genotypes were further confirmed by sequencing.
A.
Figure 3A. Sequence chromatogram of the patient line with heterozygous mutation AGC/GGC and a representative homozygous wildtype clone. The yellow box highlights the heterozygous mutation while the green box highlights the correction to homozygous wild type sequence (GGC) in the selected clone.
B.
Figure 3B. Sequence alignment of representative homozygous clone (WT) and the mutation (the patient line). A silent mutation (GCA; yellow) was introduced in the donor sequence to prevent repeated recognition and cutting by the Cas9/gRNA complex at the modification site. The mutation correction is highlighted in green.
Support Materials
When should I place my order for fresh cells?
Place your order 1-2 weeks in advance. ASC will recruit the donor that best fits your project, collect the sample, and deliver your final product within 24 hours of procurement. Same-day delivery is available to Bay Area customers, and overnight and international shipping is available to all customers. Contact us for more information.
When should I place my order for frozen cells?
1-2 days prior to the day you need the cells.
What quality control testing is conducted?
All ASC primary cell products are tested for viability and cell count. ASC guarantees 80-90% cell viability and a cell count ≥ to the total number of cells that are purchased after freeze and thaw.
Custom In Vivo Assay Services