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Updated: Dec 6, 2025
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Sensitive detection of Cre-mediated recombination using droplet digital PCR reveals Tg(BGLAP-Cre) and Tg(DMP1-Cre)
Krishnakali Dasgupta1, Samantha Lessard2, Steven Hann2
1Orthopedic Research Laboratories, Department of Orthopedic Surgery, Boston Children's Hospital, Boston, MA, United States of America; Department of Genetics, Harvard Medical School, Boston, MA, United States of America.
Abstract:
In humans, somatic activating mutations in PIK3CA are associated with skeletal overgrowth. In order to determine if activated PI3K signaling in bone cells causes overgrowth, we used Tg(BGLAP-Cre) and Tg(DMP1-Cre) mouse strains to somatically activate a disease-causing conditional Pik3ca allele (Pik3caH1047R) in osteoblasts and osteocytes. We observed Tg(BGLAP-Cre);Pik3caH1047R/+ offspring were born at the expected Mendelian frequency. However, these mice developed cutaneous lymphatic malformations and died before 7 weeks of age. In contrast, Tg(DMP1-Cre);Pik3caH1047R/+ offspring survived and had no cutaneous lymphatic malformations. Assuming that Cre-activity outside of the skeletal system accounted for the difference in phenotype between Tg(BGLAP-Cre);Pik3caH1047R/+ and Tg(DMP1-Cre);Pik3caH1047R/+ mice, we developed sensitive and specific droplet digital PCR (ddPCR) assays to search for and quantify rates of Tg(BGLAP-Cre)- and Tg(DMP1-Cre)-mediated recombination in non-skeletal tissues. We observed Tg(BGLAP-Cre)-mediated recombination in several tissues including skin, muscle, artery, and brain; two CNS locations, hippocampus and cerebellum, exhibited Cre-mediated recombination in >5% of cells. Tg(DMP1-Cre)-mediated recombination was also observed in muscle, artery, and brain. Although we cannot preclude that differences in phenotype between mice with Tg(BGLAP-Cre)- and Tg(DMP1-Cre)-mediated PIK3CA activation are due to Cre-recombination being induced at different stages of osteoblast differentiation, differences in recombination at non-skeletal sites are the more likely explanation. Since unanticipated sites of recombination can affect the interpretation of data from experiments involving conditional alleles, we recommend ddPCR as a good first step for assessing efficiency, leakiness, and off-targeting in experiments that employ Cre-mediated or Flp-mediated recombination.
Insights
Activating PIK3CA mutations in bone cells can cause overgrowth, but off-target recombination in non-skeletal tissues leads to unexpected phenotypes. Droplet digital PCR (ddPCR) is recommended for assessing recombination in conditional mouse models.
Area of Science:
- Molecular Biology
- Genetics
- Developmental Biology
Background:
- Somatic activating mutations in PIK3CA are linked to skeletal overgrowth in humans.
- PI3K signaling plays a critical role in bone cell function and development.
Purpose of the Study:
- To investigate if activated PI3K signaling specifically in bone cells causes skeletal overgrowth.
- To determine the impact of PIK3CA activation in osteoblasts and osteocytes on mouse development.
- To assess the extent of Cre-mediated recombination in skeletal and non-skeletal tissues using different Cre driver lines.
Main Methods:
- Utilized Tg(BGLAP-Cre) and Tg(DMP1-Cre) mouse strains to activate a conditional Pik3caH1047R allele.
- Developed sensitive droplet digital PCR (ddPCR) assays to quantify recombination rates.
- Analyzed phenotypes including survival, malformations, and tissue-specific recombination.
Main Results:
- Tg(BGLAP-Cre);Pik3caH1047R/+ mice exhibited cutaneous lymphatic malformations and premature death.
- Tg(DMP1-Cre);Pik3caH1047R/+ mice survived without lymphatic malformations.
- Tg(BGLAP-Cre) showed significant recombination in non-skeletal tissues (skin, muscle, artery, brain), including CNS regions.
- Tg(DMP1-Cre) also showed recombination in non-skeletal tissues, but potentially to a lesser extent or different pattern.
Conclusions:
- Off-target Cre-recombination in non-skeletal tissues is a likely explanation for the differing phenotypes observed.
- Unanticipated recombination sites can complicate the interpretation of conditional allele experiments.
- ddPCR is a valuable tool for evaluating recombination efficiency, leakiness, and off-targeting in Cre/Flp-mediated recombination studies.
