Application of droplet digital PCR in the analysis of genome integration and organization of the transgene in BAC transgenic mice

Abstract

Transgenic (Tg) mice containing bacterial artificial chromosome (BAC) DNA are widely used for gene expression analysis and gene therapy models because BAC transgenes provide gene expression at physiological levels with the same developmental timing as endogenous genes. To ensure correct interpretation of transgene functions, investigation of the genomic organisation and integration of the BAC transgene is required. Here, we describe a reliable method based on droplet digital PCR (ddPCR) and inverse PCR to estimate copy number, genomic organisation and insertion sites of BAC transgenes in the mouse genome. We generated BAC Tg mice containing fragments of BAC clone RP23-59P20. ddPCR and iPCR analysis showed that the transgene consisted of five fragments of the BAC clone containing the Mkrn3 gene region, and that the transgene was inserted into Bckdhb, homozygous deletion of which causes the maple syrup urine disease phenotype. The ddPCR method described here should prove useful for analysis of genomic organisation and integration of BAC transgenes.

Development of an AAV9-RNAi-mediated silencing strategy to abrogate TRPM4 expression in the adult heart

“…Droplet digital PCR (ddPCR) was carried out to determine the AAV genome copy number per host genome using the Magel2 gene as a diploid reference [ 28 ]. Primers and an FAM-labeled probe specific for the bGHpA (bovine growth hormone polyadenylation signal) sequence were used to determine the AAV genome copies (Table S2 ).…”

Preclinical evaluation of stereopure antisense oligonucleotides for allele-selective lowering of mutant HTT

“… 39 Because the copy number, integrity and expression of BAC transgenes can vary, with some BAC inserts comprising partial transgenes and with the potential for transgene copy number to change through generations, 62 , 63 , 64 we evaluated the copy number in our BACHD colony ( Figure S3 B) using droplet digital PCR (ddPCR). 65 The copy number varied across the transgene, with the 5′ region containing the glutamine-encoding repeat and the pan-targeting sequence at an estimated 13 copies, whereas the SNP3-containing region of the transgene was present at an estimated nine copies, indicating that while all copies of the transgene contain the pan-targeting sequences, not all copies of the transgene contain SNP3 ( Figure S3 B). Although the mouse Htt gene is present in this model, mouse Htt does not contain SNP3.…”