title: Loss of the RING ligase praja2 boosts tyrosine kinase receptors in clear cell carcinoma
design: Loss of the RING ligase praja2 boosts tyrosine kinase receptors in clear cell carcinoma
library_construction_protocol: LoxP (L83) site and a FNFL (Frt- Neo- Frt-LoxP) cassette were engineered to flank exon 2/3 (2 Kb) of the praja2 allele to generate �floxed/neo� praja2 on a Bacterial Artificial Chromosome (BAC). A gene targeting vector was constructed by retrieving the 2 kb short homology arm (5� to L83), the floxed sequence containing exon 2/3, the FNL cassette, and the 5 kb long homology arm (end of FNFL to 3�) into a plasmid vector carrying a DTA (Diphtheria Toxin Alpha chain) negative selection marker. The FNFL cassette confers G418 resistance during gene targeting in KV1 (129B6 hybrid) ES cells and targeted ES cells were confirmed by PCR and southern blotting analysis. One of the ES positive clones was injected into C57BL/6J blastocysts to generate chimeric mice. Chimeric founder male mice were bred with C57BL/6J females for germline transmission and the consequent generation of a mouse line carrying the genetic locus of praja2 under the control of loxP sites. (Authorization number: prot. 2014/0015838). After the deletion of Neo Cassette by Flpe recombinase, praja2 floxed mice were crossed with Hprt-Cre mice for the excision of exon 1 and 2. The resulting heterozygous praja2 KO mice (praja2+/-) were backcrossed into the C57BL/6 J background for three generations (F3). Praja2+/- mice were then interbred to produce homozygous praja2-/- animals. Genomic DNA was extracted as described previously[73] by ear clip biopsies and KO mice were genotyped by PCR using the following primers: FNFFw, 5�- GATGTTCCAAAGGAGAACACCTCAG-3�; FNFRw, 5�-TATGCAGGAAAAACACACTC GGTTC-3�; LNLFw, 5�-GAAAGGGTTGGTTTGGAGTACAGGT-3�. The wild type fragment (466 bp) was generated by the primers FNFFw and FNFRw; the KO fragment (320 bp) was generated by the primers LNLFw and FNFRw. Amplifcation conditions were: 95 �C for 5 min; 35 cycles: 95 �C for 30 s, 62 �C for 30 s, 72 �C for 1 min; 72 �C for 10 min. Animals were group housed (five per cage), at a constant temperature (22�1 �C) on a 12 h light/dark cycle (lights on at 7 AM) with food and water ad libitum. (Autorization n. 924/2017-PR del 29/11/2017) RNAs were extracted from FFPE-embedded tissues using RNeasy FFPE kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA purity was assessed by NanoDrop� 2000/2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), whereas its concentration and integrity were measured by Qubit fluorimeter using RNA assay kit (both Life Technologies, Carlsbad, CA, USA) and Agilent 4200 Tapestation System (Agilent, Santa Clara, CA, USA), respectively. Indexed libraries were prepared starting from 500 ng of total RNA using TruSeq Stranded Total RNA preparation kit (Illumina Inc., San Diego, CA, USA).