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Generating a Transgenic Mouse Line Containing the Plasma Membrane Phosphatidylinositol 4,5‐Bisphosphate Depletion System
Author(s) -
Gulyás Gergő,
Szalai Bence,
Geiszt Miklós,
Balla Tamás,
Hunyady László,
Várnai Péter
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.946.4
Subject(s) - transgene , phosphatidylinositol , microbiology and biotechnology , biology , phosphatidylinositol 4,5 bisphosphate , kinase , biochemistry , chemistry , gene
Phosphoinositides are key regulators in many signaling cascades and several cellular processes. These lipids are located in cellular membranes containing different forms of these lipids. The plasma membrane (PM) is enriched in two phosphorylated variants, phosphatidylinositol 4‐phosphate and phosphatidylinositol 4,5‐bisphosphate (PIP2). To discover the role of these lipids, several applications have been developed. One of them is the rapamycin inducible heterodimerization system, based on the rapid and irreversible interaction of the FRB and FKBP12 proteins. In our system FRB was targeted to the PM using the localization sequence of the Fyn tyrosine kinase, whereas the FKBP was fused to an enzymatically active cytoplasmic 5‐phosphatase. After rapamycin treatment, the enzyme recruited to the PM, where rapid depletion of PIP2 could be achieved. Our goal was to generate a genetically modified mouse line containing this lipid depletion system, which can be highly capable to investigate the physiological role of PIP2 in primary prepared cells or in intact tissues and organs. Our plasmid used for the transgenic application contained both protein coding sequences of the depletion system in the same vector. To reach the stochiometric expression of the two distinct fragments, a viral T2A sequence was inserted between the protein coding sequences. For further detection of the transgene the PM targeted protein was fluorescently tagged. The transgenic construct was driven by a CMV promoter to reach a general expression in various animal tissues. The transgenic modification was carried out by applying the Sleeping Beauty transposase in FVB/Ant mouse strain. Based on the genotyping data of the founder generation, two distinct lines with low and high copy number were selected for the further investigations. Interestingly, transgene showed detectable expressions only in pancreatic acinar cells and in keratinocytes. To investigate the functional effect of the PIP2 depletion we carried out cytoplasmic calcium ion measurements. Using freshly prepared pancreatic acinar cells of the F2 generation rapamycin treatment did not resulted a reduced cytoplasmic calcium signal upon stimulation the cells with carbachol, indicating the low expression level of the PIP2 depletion system. A mathematical model was created, which predicts of the increase of protein expression, so we decided to select the animals with the highest copy numbers in each generation and use them as parents of the further generations. The efficiency of lipid depletion is monitored by measuring the translocation of PLCdelta1‐PH‐domain based PIP2 sensors, as well as by measuring other PIP2‐dependent cellular responses. Support or Funding Information National Research, Development and Innovation Fund (NKFI K105006)