In this report, we describe two new alleles of Pax2 created by inserting the enhanced green fluorescent protein coding region into the 5′ untranslated leader sequence.
GLTP-siRNAs were produced using pSuper.retro.puro vector (gift of Drs. Albert Bendelac & Yuval Sagiv), which we modified further by inserting the enhanced green fluorescent protein ORF at the C-terminus of the puromycin N-acetyl transferase coding sequence (pSuper.puro-egfp).
For that purpose, we generated P. patens knock-in lines by inserting the Citrine yellow fluorescent protein gene [ 47] just before the stop codon of PpHsp16.4 by means of homologous recombination.
Testing of this plasmid vector for enhanced fluorescence screening was conducted by inserting the PCR fragment encoding the fluorescent protein hcRed (between MCS1 and MCS2) [10].
The plasmids for overexpression of EYFP, EYFP-D70-SF-1 and 3-FLAG-SF-1 were constructed by inserting the coding sequence of enhanced yellow fluorescent protein (EYFP), or wild-type or N terminal truncated (D70) SF-1 into pAS4w.1.Pbsd, which contains seven copies of modified tetO sequence.
Both wild-type and mutant cortactin proteins were tagged with green fluorescent protein (GFP) by inserting the cortactin cDNAs into pEGFP-N1 plasmid (Clonetech); the cortactin-GFP sequences were subcloned into pCS2+ vector [19] and then into pcDNA3.1.
On the other hand, HIV particles, which themselves were fluorescent due to the incorporation of a fluorescent fusion protein, were generated by inserting the gfp gene between the matrix and capsid Gag sequences [ 8].
AAV vectors harboring reporter genes were constructed by inserting the genes encoding Gaussia luciferase (Gluc) or enhanced green fluorescent protein (EGFP) between the cytomegalovirus (CMV) promoter and bovine growth hormone polyA (BGH polyA) in pAAV2neo (Fig. 1A) or pAAV5neo [14].
The GFP (green fluorescent protein)–PRH plasmid was constructed by inserting the PRH cDNA from a BlueScipt clone into the EcoRI and KpnI sites of eGFPc1 (Clontech).
The expression plasmid for enhanced green fluorescent protein (EGFP) (CLONTEC H Laboratories CA, USA) was constructed by inserting the cDNA into pcDNA3.1+ to produce pcDNA-EGFP.
