Turboblotter

The linearized Plin2-Flox-Neo vector was electroporated into 129/svJEv ES cells. ES cell clones with the desired homologous recombination event were identified by Southern blotting using Turbo blotter (Schleicher & Schuell) followed by hybridization of membranes with [α-³²P]dCTP (PerkinElmer, Wellesley, MA) radiolabeled probes generated with the Megaprime DNA labelling System (Amersham Biosciences). Targeting efficiency was 10 positive events of 96 screened clones.
Animal use for generation of the floxed Plin2 model was registered with and approved by the NIDDK Animal Care and Use Committee (plan #K039-LCDB-10) and followed the national Guide for the Care and Use of Laboratory Animals. Positive ES cells were microinjected into blastocysts derived from C57BL/6J females and transferred to pseudopregnant recipients. Chimeric offspring were mated with C57BL/6J mice for germline transmission. The presence of the targeted allele in the agouti-colored offspring was confirmed by PCR and Southern blot hybridization. Floxed Plin2 mice (Plin2^flox-Neo) were mated with mice expressing MMTV-Cre recombinase to obtain mice with global deletion of Plin2 (Plin2^−/−) mice, which were confirmed for deletion of exons 3, 4, and 5 by PCR, RT-PCR, and Southern blot hybridization (see Supplemental Fig. S1).

Total RNA (5 µg) were run on denaturing agarose gels containing 0.25 M formaldehyde, and the gels were stained with ethidium bromide (EtBr) to visualize 23S and 16S rRNA. The fractionated RNA was transferred to nylon membranes (Schleicher & Schuell, Germany) using a Turboblotter (Schleicher & Schuell, Germany). The mRNA levels were determined by hybridizing the membrane with a gene specific, ³²P-labeled probe (Takara, Japan) prepared by PCR amplification with their respective primer pair as indicated in Table S5. Autoradiography was conducted using an IP plate (Fujifilm, Japan) and a Multiplex Bio-Imaging system (FLA-7000; Fujifilm, Japan).