Molecular Genetics

2.4.1 Molecular cloning

All 5’ cloning and sub-cloning primers included a CCACC Kozak recognition site before the start codon; they also include restriction enzyme recognition sites as required by the vector. The first cDNA strand was synthesised using an Oligo dT primer (Sigma-Aldrich) and SuperScriptTM II or SuperscriptTM III (Life Technologies) as RNA-dependent DNA polymerase. Gene-specific DNA was PCR amplified using mRNA-specific primers of interest through 35 cycles with either Taq polymerase (Qiagen, Hilden, Germany) (mRNA < 800 b.p.) or Pfu polymerase (Agilent, Santa Clara, CA, U.S.A., or Promega, Madison, WI, U.S.A.). DNA was then purified (Wizard® SV, Promega or PCR purification kit, Macherey- Nagel) and digested with the appropriate restriction enzymes (Promega or NEB). Digested DNA fragments were run on an agarose gel (1% w/v) and gel eluted (Wizard® SV, Promega). Target vectors and inserts were cut with compatible restriction enzyme sites and purified. The vectors were 5’-dephosphorylated using Antarctic phosphatase (NEB). Ligation was conducted on all insert-vector pairs using a 1:3 to 1:5 insert:vector ratio (Quick ligase, NEB). A list of all primer sequences and specifications used for molecular cloning can be found in Appendix C. All expression vector maps are shown in Appendix D. The gel-eluted DNA was precipitated with the help of yeast tRNA and dissolved in MilliQ H2O.

Molecular cloning of murine transporters B0AT1 and B0AT3, ancillary proteins collectrin, and ACE2 has been previously reported [37, 169, 177]. . Mouse small intestinal total RNA was isolated as a PCR template to amplify mouse APN cDNA (RNeasy plus, Qiagen or

79 Nucleospin® RNA II, Macherey-Nagel, Düren, Germany). EcoRI endonuclease restriction sites were added to both the sense and anti-sense cloning primers to facilitate sub-cloning. Following amplification of APN, the PCR product was purified from an agarose gel (1% w/v) (Wizard® SV, Promega) and inserted into pZero Blunt (Life Technologies), using the same procedure as described below for all direct cloning. The insert was then excised using EcoRI and ligated into pGem-He-Juel (Appendix D, abbreviated pGHJ from here on) for in vitro transcription.

Molecular cloning of murine syntaxin 1A or syntaxin 3 and 7 was conducted using total RNA isolated from mouse brain or mouse kidney as a template, respectively (RNeasy plus, Qiagen). For syntaxin 1A and 7, HindIII restriction endonuclease sites were incorporated in both the 5’ and 3’ cloning primers. For syntaxin 3, EcoRI restriction endonuclease sites were added to the 5’ and 3’ ends of the cloning primers. Syntaxin 1A was first inserted by blunt-end ligation into the pCRTM-TOPO XL® vector using Vaccinia virus topoisomerase I (Life Technologies) and the same procedure as described below for all direct cloning. The insert was then excised using HindIII or EcoRI and sub-cloned into pGHJ.

Human ACE2 and mouse collectrin truncation primers were designed based on multiple pairwise alignments of ACE2 and collectrin. ACE2 ∆18-580 retains the collectrin homology consensus sequence, beginning just upstream of the consensus start site (ACE2-591). ACE2

∆18-500 was designed to retain an in silico identified tyrosine-based ER sorting signal (ELM Accession ELME000120) annotated at ACE2-510-513, which may be important for trafficking to the plasma membrane. The predicted N-terminal signal peptide (ACE2 residues 2-18, collectrin residues 2-14) was retained in all constructs. All collectrin truncation

80 boundaries were determined using information garnered from the collectrin sequence analysis and bioinformatics (section 2.7.4).

For CHO cell experiments, syntaxin 1A and syntaxin 3 were sub-cloned from pGHJ vectors into pcDNA3.1+ mammalian expression vector using the same HindIII (syntaxin 1A) and EcoRI (syntaxin 3) endonuclease sites that were used for the original cloning. Sub-cloning was verified by restriction digests and sequencing. All pcDNA3.1+ syntaxin constructs were isolated using an endotoxin-free midi DNA preparation (NucleoBond® Xtra EF Plus, Macherey-Nagel) before transfection into mammalian cell cultures.

Ligated constructs were introduced into E.coli XL-1 cells by electroporation or by chemical transformation into E.coli DH5α or DH10β strains (NEB). After transformation bacteria were incubated in SOC media for 1.5 hrs at 37°C. Following recovery, serial dilutions of cells were spread onto LB-agar plates (Greiner Bio-One, Kremsmünster, Austria) containing either ampicillin (100 μg/ml) or kanamycin (50 μg/ml) (Sigma-Aldrich) depending on the antibiotic resistance gene used as a selection marker. The plasmid size of transformed over-night colonies was verified using rapid colony screening. To this end, individual colonies were streaked onto fresh antibiotic selection plates and picked following 12-18 hrs incubation. Bacteria were lysed using colony cracking buffer, resolved in resuspension buffer and the extract run on a 1% (w/v) agarose gel together with a supercoiled DNA marker (NEB or Takara, Shiga, Japan). Correctly transformed colonies were propagated using the same selection antibiotics and concentrations in Luria-Bertani Broth (AMRESCO, Solon, OH, U.S.A.), followed by plasmid isolation using mini-, midi-, or maxi-preparations (Macherey- Nagel, or Promega).

81 Monocarboxylate-transporter fusion constructs rMCT1-B0AT1 and rMCT1-B0AT3 were generated using overlap extension PCR on cDNA templates. The rMCT1 sense primer was designed to incorporate a XbaI cutting site before the start codon. The B0AT1 antisense primer comprised a XbaI site, while the B0AT3 antisense primer comprised an EcoRI site. The overlap primers contained 21bp from each cDNA in either sense or antisense orientation excluding stop and start codons.

The final integrity and orientation of the cloned cDNA was verified by sequencing (Australian National University, Biomolecular Resource Facility). All genes, cRNA and proteins in this thesis are of homo sapiens or mus musculus origin unless indicated otherwise.

2.4.2 Site-directed mutagenesis

Single point mutations were introduced into B0AT1, B0AT3, APN and collectrin cDNA using the QuickChange® II or QuickChange® Lightning Site-Directed Mutagenesis Kits (Stratagene, La Jolla, CA, U.S.A.). Complementary primers containing the desired mutation flanked by 15-20 nucleotides corresponding to the cDNA sequences of target proteins were used to amplify the complete cDNA-containing pGHJ vector. Following 16 rounds (single b.p. change) or 18 rounds (multiple b.p. changes) of PCR amplification, reactions were treated with DpnI, which only recognises methylated DNA, thereby digesting the template DNA but not the amplified, mutated PCR product. Mutant plasmids were introduced via electrical transformation into E.coli XL-1 cells or chemical transformation of E.coli 5Hα or 10Hβ strains (NEB). Transformed colonies were grown on LB-agar plates (Greiner) containing ampicillin (100 μg/ml) over night. Plasmid DNA was isolated from single colonies grown in LB-media (AMRESCO) overnight using mini-preparations (Macherey-

82 Nagel, or Promega). All mutations were verified by DNA sequencing (Australian National University, Biomolecular Resource Facility).

2.4.3 RNAi knockdown and reverse transcription PCR

Small interfering RNAs for CHO syntaxin 1A and 3 were selected from mouse MISSION predesigned siRNA (Sigma-Aldrich) binding to sequence regions that were 100 % identical to the corresponding Chinese hamster sequences. CHO-SLC6A19-Collectrin cells were transfected with the RNAi pairs when 30-50 % confluent using with 50 pmol of RNAi/condition (lipofectamine® RNAiMAX, Life Technologies) and incubated at 37 °C for a further 48 hrs before experimental use. To verify silencing, total RNA was extracted from transfected cells (Macherey-Nagel, NucleoSpin® RNA) and 1 µg was reverse transcribed into cDNA (Superscript® II, Life Technologies). For semi-quantitative analysis, transcripts were amplified for 25 cycles. Following RT-PCR, samples were loaded onto a 1 % (w/v) agarose gel, and quantified using U.V. densitometry after running and staining (SYBR® safe, Life Technologies).