Pcr2.1 plasmid

A genomic segment of MFSD8 spanning exon 5 (hg19, chr4:128864292‐128865864) was amplified from patient DNA using primers with NotI and BamHI recognition sequences (Table S5). PCR products were cloned into the pCR2.1 plasmid (Invitrogen‐Life Technologies, Carlsbad, California). Wild type (WT) and mutant inserts were excised by digestion and subcloned into digested pSPL3_2096 (Invitrogen‐Life Technologies, Carlsbad, California). HEK293T cells were transfected with pSPL3‐MFSD8 constructs using Lipofectamine (Invitrogen‐Life Technologies, Carlsbad, California). Total RNA was extracted after 24 hours (peqGOLD Total RNA Kit, VWR, Radnor, Pennsylvania) and reversely transcribed (Transcriptor High Fidelity cDNA Synthesis Kit, Roche Applied Science, Penzberg, Germany). The cDNA was PCR amplified with pSPL3 exon primers and Sanger sequenced (Applied Biosystems, Foster City, California).

Antisense and sense probes for human prostacyclin mRNA were prepared as follows. A polymerase chain reaction (PCR) fragment (420 base pairs) generated the amplification of mesangial mRNA with the primer pair depicted below that was ligated and cloned in a pCR 2.1 plasmid (Invitrogen, USA). The following primers were used to generate a murine-PGIS riboprobe: forward-GGCTGGCTGGGTTGAGAATC and reverse-GACCGTGCGAAGGTTGGTAT. Cloned cDNA fragments were sequenced according to the dideoxy method to confirm the identity and orientation of the inserts. In situ hybridization was performed as described previously [16 (link)]. After development in Kodak D-19, slides were counterstained with hematoxylin. Photomicrographs were taken with a Zeiss Axioskop microscope using bright field optics.

RNA extractions from the serum samples were performed as previously described [21 (link),22 (link)]. Real-time PCR for type 1 and type 2 PRRSV, and vaccine strain were used to quantify PRRSV genomic cDNA copy numbers using RNA extraction from serum samples. The sequences of primers and probes in real-time PCR are 100% complementary to the sequences of the challenge viruses (except 89.5% complementary for reverse primer of type 2 PRRSV). Real-time PCR was considered positive if the cycle threshold (C_T) level was obtained at ≤ 45 cycles.
To construct a standard curve, real-time PCR was performed in quadruplicate in two different assays: (i) 10-fold serial dilutions of the PRRSV plasmid were used as the standard, with concentrations ranging from 10¹⁰ to 10³ copies/mL and (ii) 10-fold serial dilutions of the challenging type 1 and type 2 PRRSV cultured in alveolar macrophages and MARC-145 cells, respectively, from 1.0 × 10⁶ TCID₅₀/mL to 1.0 × 10^-1 TCID₅₀/mL. The PRRSV plasmid was prepared as previously described [23 (link)]. Briefly, the transcript cDNA product was cloned into the pCR2.1 plasmid (Invitrogen, Carlsbad, CA, USA). The recombinant plasmid was purified using a plasmid Miniprep kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions, and the concentration of the purified plasmid was determined using a spectrophotometer.