Pgem t easy

To construct binary construct pMU2T-bar-RS2 we first built a 2 T-DNA binary vector for the cloning of expression cassettes by amplifying T-DNA borders using PCR and clone them into the binary vector pPZP201 (Hajdukiewicz et al., 1994 (link)). To do this two PCR reactions were done to amplify the borders using two sets of primer pairs (Table S1), and the same template was amplified for one left border. After amplification, both PCR products were cloned into pGEM-T Easy (Promega, Madison, USA) and two right borders from pGEM-T Easy were digested using PstI and SphI, and ligated into PstI and SphI sites of pPZP201. Afterwards the left border in pGEM-T Easy was cut with AatII and SphI, and further cloned into the vector with the two right borders. This two T-DNA vector resulted in pMU-2T. The RNAi expression cassette digested from pMU103 (Flores et al., 2008 (link)) and then cloned into the first T-DNA region in pMU-2T. Then, a bar expression cassette was PCR-amplified from pZY102 (Zeng et al., 2004 (link)) as EcoRI-PstI (blunt-ended) fragment and cloned into EcoRI/SacI (blunt-ended) sites of the second T-DNA region in a reverse orientation. Agrobacterium-mediated soybean transformation followed our improved protocol derived from previous one (Zeng et al., 2004 (link); Flores et al., 2008 (link)) using an elite soybean genotype “Maverick.”

The mGDNF construct with deleted pre- and pro-regions and with an EGFP tag was generated by introducing a HindIII site, a Kozak sequence, and an extra start codon upstream of the “m” part as well as by removing the stop codon and introducing a BamHI site in the 3′ region of mGDNF using PCR. The following primers were employed: Gdnf^HindIII(F) 5′-AAGCTTCCACCATGTCACCAGATAAACAA-3′ and Gdnf^BamH1(R)5′-GGATCCCAG ATACATCCACACCTTTTAGCGG-3′. The plasmid pGEM-T Easy (Promega) containing the full-length human GDNF cDNA [25 ] was used as the template. PCR was performed using the Tersus polymerase (Evrogene) and the following program: 94 °C for 1.5 min; 25 cycles of 94 °C for 15 s, 57 °C for 20 s, and 72 °C for 15 s; and final 72 °C for 10 min. The resulting 354 bp (118 amino acids) fragment was isolated from agarose gel using a Qiaquick Gel Extraction Kit (Qiagen) and cloned into pGEM-T Easy (Promega). The HindIII/BamHI fragment of the resulting construct pGEM/mGdnf was cloned into the corresponding sites of pEGFP-N1 (Clontech). For the control we used construct with pre-pro-GDNF, which were prepared using the primers T3 (F) 5′-ATTAACCCTCACTAAAGGGA-3′ и Gdnf ^BamH1 5′-TGGATCCCAGA TACACCACACCTTTTAGCGG-3′. This construct was obtained according to the protocol described elsewhere [1 (link)].

Single whole-mount in situ hybridisation was performed as described [31 (link)]. Embryos were photographed with a LEICA Z6 APO stereoscope coupled to a LEICA DFC490 camera.
Digoxigenin-labelled antisense RNA probes were synthesised from DNA templates of deltaC [32 (link)], her1 [33 (link)], her7 [34 (link)], mespb [35 (link)], pcdh8 [36 (link)], myod1 [37 (link)], cb1045 [38 (link)], spaw [39 (link)], pitx2 [40 (link)], myl7 [41 (link)], cxcl12b [25 (link)], etv2 [42 (link)], foxj1b [19 (link)], foxc1b [43 (link)], dmrt2a [4 (link)], cyp1a, which was cloned in pGEM-T Easy (Promega) using the pF-cyp1a (5’-ATGGCTCTGACTATTCTTCCAATATTGGG-3′) and pR-cyp1a (5’-CTAGAACCCAGGCTGTGGTGTGACCCGA-3′) and pxdc1b, which was cloned in pGEM-T Easy (Promega) using the pF-pxdc1b (5’-ATGGCATCGGCGATTTTTGAGGGCA-3′) and pR-pxdc1b (5’-AAGTCAGTTTCAAAAGGAACCAGA-3′).
For HA immunohistochemistry, the embryos were fixed after heat-shock in 4% paraformaldehyde overnight, incubated with rat anti-HA antibody (1:200, 3F10, Roche #11867423001) followed by anti-rat Alexa Fluor 594 (1:500, Thermo Fisher #A11007). F-actin and nuclei were detected with Alexa Fluor 488-Phalloidin (1:400, Thermo Fisher #A12379) and DAPI (10 μL.mL^− 1), respectively. Embryos were photographed with a Zeiss LSM 510 Meta.

Primers were designed based upon regions of target genes conserved in multiple mammalian genomes. Appropriate primer sets were used to amplify fragments of badger cytokine or TLR genes from PBMC cDNA using Myfi PCR mix (Bioline), according to manufacturer’s instructions. Products were cloned into pGEM TEasy (Promega) and sequenced using plasmid targeting (M13) or gene specific primers with BigDye chemistry (Life Technologies), according to manufacturer’s instructions. Sequences were analysed using Bioedit58 , and badger specific sequence used to design primers for 5′RACE PCR. 5′RACE cDNA was generated using a SMARTer RACE kit (Clontech) according to manufacturer’s instructions. PCR products generated from RACE PCR products were cloned into pGEM TEasy, as described, or into pTarget (Promega) if longer than 1 kb, and sequenced as above (European Nucleotide Archive accession numbers are given in Supplementary Table 1). Sequences were analysed using BioEdit and MEGA59 (link) and compared to human, mouse dog and ferret sequences to identify target regions for intron spanning QRT-PCR assays.

The qPCR product was treated following the protocol described by Sambrook and Russel (2001). The DNA fragment was purified from an agarose gel (2.5%, w/v) using the QIAGEN GmbH purification kit. The amplified fragment was then cloned into the pGEM-T easy plasmid (Promega) for the cloning of qPCR amplicons from P. putida U cDNA transcripts.
Transformation of chemically competent cells from the E. coli DH10B strain was carried out using the RbCl method [48 (link)]. Plasmid extraction from the E. coli DH10 B-derived strains was performed using the QIAGEN GmbH miniprep (Antwerp, Belgium) extraction kit. Plasmid concentration and purity were determined using a Nanodrop model 6345 spectrophotometer from Thermo Fisher.
Finally, selected plasmids were sequenced using universal m13 primers flanking the multiple cloning sites from pGEM-T easy (Promega). Sanger sequencing of the different plasmids was performed by Secugen S.L. (Madrid, Spain). Sequences from the amplicons determined in this work (Figure S1) from P. putida U genomic DNA have been deposited in GenBank under accession numbers spuC1 (OR435851), spuC2 (OR435852), gabT1 (OR435853), gabT2 (OR435854), rpoD (OR435855), rpoN (OR435856), and recA (OR435857).

Escherichia coli strain DH-5α was used for plasmid manipulations and propagation of pGEM-Teasy (Promega, Madison, WI, USA). pCAMBIA2301 vector² was modified by removal of the 35S promoter driven uidA expression cassette. AtMTP1 (At2g46800) and AtZIP1 (At3g12750) genes were PCR-amplified from A. thaliana, cloned into pGEM-Teasy vector (Promega) and verified by sequencing. A total of five gene constructs were generated for integration into cassava. The PAT:AtMTP1 expression cassette in which the tuber specific class I patatin promoter from potato (Rocha-Sosa et al., 1989 (link)) was used to drive expression of AtMTP1, and the PAT::AtZIP1 expression cassette consisted of the patatin promoter driving AtZIP1(At3g12750). In addition, the FMV promoter was fused to AtMTP1 and AtZIP1 to generate FMV:AtZIP1 and FMV:AtMTP1, respectively. A final construct was produced carrying both zinc genes within the same region of the T-DNA to make PAT:AtZIP1- PAT:AtMTP1.
All pCAMBIA2301-based transformation vectors were mobilized into Agrobacterium tumefaciens strain LBA4404 by electroporation and used for delivery of T-DNA into plant cells. Friable embryogenic callus (FEC) of cultivar 60444 was produced and used for the production and recovery of transgenic plants as described by Taylor et al. (2012) (link).