Table 3.
Genes cloned for overexpression in a HEK293T system
Single cells identified as astrocytes on the basis of established marker genes (19) were analyzed to identify transcripts fulfilling the following criteria. First, transcripts had to be present in ≥90% of identified astrocytes. Second, transcripts had to encode proteins containing a predicted transmembrane region. Third, only proteins localizing to the plasma membrane were considered. Only proteins fulfilling these criteria could realistically be used in the immunoisolation of live (non-fixed) astrocytes. This table lists identified transcripts fulfilling these criteria and the corresponding PubMed accession numbers of the transcripts used to design subsequent cloning steps. Essentially, PCR primers were designed to flank ORFs of genes of interest (capital letters) as well as to incorporate unique restriction sites on both ends (lowercase letters). Sequences were then amplified by PCR before ligation into the pCAGIG plasmid. Plasmids were verified by sequencing and checked against the deposited amino acid sequence for the respective proteins. Single-nucleotide polymorphisms that did not affect the amino acid sequence were tolerated. In addition to encoding genes of interest, these constructs also encoded cytosolic GFP to act as a marker for cell transfection.
| Gene (PubMed accession number) | Protein (PubMed accession number) | Primers | Restriction sites |
|---|---|---|---|
| Slc1a2 (NM_001077514.3) | Excitatory amino acid transporter 2 (GLT-1) (NP_001070982.1) | Forward, taagtatcGAATTCATGGCATCAACAGAGGGTG; reverse, taagtatcCTCGAGTTATTTTTCACGTTTCCAAGGTTCT | EcoRI/XhoI |
| Slc1a3 (NM_148938.3) | Excitatory amino acid transporter 1 (GLAST) (NP_683740.1) | Forward, taagtatcCTCGAGATGACCAAAAGCAACGGAGAAG; reverse, taagtatcGCGGCCGCCTACATCTTGGTTTCGCTGTCT | XhoI/NotI |
| Atp1a2 (NM_178405.3) | Na+/K+-transporting ATPase subunit α-2 precursor (NP_848492.1) | Forward, taagtatcCTCGAGATGGGTCGTGGGGCAG; reverse, taagtatcGCGGCCGCTCAGTAGTACGTCTCCTTCTCC | XhoI/NotI |
| Atp1b2 (NM_013415.5) | Na+/K+-transporting ATPase subunit β-2 (NP_038201.1) | Forward, taagtatcCTCGAGATGGTCATCCAGAAAGAGAAGAAGA; reverse, taagtatcGCGGCCGCTCAGGTTTTGTTGATCCGGAGTT | XhoI/NotI |
| Ppap2b (NM_080555.2) | Lipid phosphate phosphohydrolase 3 (NP_542122.1) | Forward, taagtatcCTCGAGATGCAAAGCTACAAGTACGACAA; reverse, taagtatcGCGGCCGCCTACACCATGTTATGGTGATTGTTC | XhoI/NotI |
| Gja1 (NM_010288.3) | Gap junction α-1 protein (NP_034418.1) | Forward, taagtatcCTCGAGATGGGTGACTGGAGCGCC; reverse, taagtatcGCGGCCGCTTAAATCTCCAGGTCATCAGGCCG | XhoI/NotI |
| Tlcd1 isoform a (isoform 1) (NM_026708.2) | Calfacilitin isoform a (isoform a) (NP_080984.1) | Forward, taagtataGAATTCATGCCCCTACTGTTCCACC; reverse, taagtataGCGGCCGCTCACTCAGTCAAGAATTTGTCTTTT | EcoRI/NotI |
| Tlcd1 isoform c (isoform 3) (NM_001291236.1) | Calfacilitin isoform c (isoform c) (NP_001278165.1) | Forward, taagtatcGAATTCATGAGATGGCCGTGGGAT; reverse, taagtatcGCGGCCGCTCACTCAGTCAAGAATTTGTCTTTT | EcoRI/NotI |