iPSC IMR90-4 ApoE Genotyping

Introduction

The ApoE4 allele of the apolipoprotein E (ApoE) gene is the greatest known genetic risk factor for Alzheimer’s disease. Further it has been linked to increased susceptibility and duration of infection. On the other hand, the ApoE3 allele has been shown to have a protective effect against severe sepsis. Importantly, ApoE has an important role in blood-brain barrier (BBB) integrity, with ApoE4 directly linked to reduced BBB function via multiple mechanisms. As such we want to develop two BBB models, a “healthy” ApoE3 and “diseased” ApoE4 model.

We have been using the commercially available human induced pluripotent stem cell (hiPSC) line, IMR90-4 (WiCell), for differentiation of hiPSCs to brain pericyte-like cells and brain microvascular endothelial cell-like cells. However, we needed to confirm these cells as ApoE3 homozygous to continue to use the cells as our “healthy” BBB model. There are two snps that determine the variant of ApoE, rs429358 and rs7412.

SNP	rs429358	rs7412
e2	TGC	TGC
e3	TGC	CGC
e4	CGC	CGC

We are looking for homozygous ApoE3, so T at rs429358 and C at rs7412 with the chromatogram showing a clear single peak at the snp location.

Methods

DNA was extracted from IMR90-4 (DNeasy Blood and Tissue Kit, Qiagen) and the APOE gene was amplified using published primers (Forward primer: 5’-AGCCCTTCTCCCC GCCTCCCACTGT-3’ and Reverse primer: 5’-CTCC GCCACCTGCTCCTTCACCTCG-3’), following the protocol published in Zhong et al. Molecular Neurodegeneration (2016) 11:2. The amplicon was purified (MinElute PCR Purification Kit, Qiagen), ran on a 2% agarose gel in TBE buffer, extracted from the gel and gel purified (PureLink® Quick Gel Extraction Kit, Invitrogen). This was then purified again (MinElute PCR Purification Kit, Qiagen) and sent for sequencing. The sequence was then compared to the known sequence via NIH BLAST.

Results

Our best results were sequencing with our reverse ApoE primer.

rs429358:

rs7412:

Conclusions

At rs429358 we have A/T, and at rs7412 we have G/C. This indicates we have an ApoE3 homozygous cell line.