Generation of transgenic lines

Generation of mpeg1.1 :loxP-DsRedx-loxP-GFP transgenic line

• mpeg1 promoter were cloned from genomic DNA by PCR.
• PCR products were purified by QIAquick PCR Purification Kit or QIAquick Gel Extraction Kit (QIAGEN) 
• All purified product were digested by restriction enzymes NotI-HF and SpeI-HF (New England Biolabs). Backbone vector pTol2 were digested by the same enzyme to create the same sticky ends.
• Purified digestion products were ligated using T4 DNA ligase (New England Biolabs) following the official protocols.
• Ligated product was introduced into competent cells and the transformed cells were grew on the LB culture dish at 37℃for 12hours.
• Target single colonies were screened by colony PCR using these primers:

M13 fwd:  5’-GTAAAACGACGGCCAGT-3’ ; M13 rev:  5’-CAGGAAACAGCTATGAC-3’

• The colony carried target plasmid was cultured in 4ml LB medium at 37℃ for 12hours.
• Plasmids were isolated by alkaline lysis and the plasmid were sequenced.
• Resulting pTol2-mpeg1.1 promoter was used as backbone vector in the next construction and was digested by restriction enzymes EcoRV-HF and XhoI (New England Biolabs).
• loxP-DsRed-loxP-GFP cassette was digested by the same enzymes from coro1a: loxP -DsRedx -loxP -GFP construct (Xu et al., 2015)
• Purified digestion products were ligated using T4 DNA ligase (New England Biolabs) following the official protocols.
• Ligated product was introduced into competent cells and the transformed cells were grew on the LB culture dish at 37℃for 12hours.
• Target single colonies were screened by colony PCR using these primers:

mpeg1.1-(-194)-FP:  5’-CCTTCAACTTAACGTTTGAACGC-3’

M13 fwd:   5’-GTAAAACGACGGCCAGT-3’

• The colony carried target plasmid was cultured in 4ml LB medium at 37℃ for 12hours.
• Plasmids were isolated by alkaline lysis and the plasmid were sequenced.
• Resulting pTol2-mpeg1.1:loxP-DsRedx-loxP-GFP plasmid was purified by QIAquick PCR Purification Kit(QIAGEN) and eluted by RNase free water.
• Adjust concentration of the plasmids to 50ng/ul 
• Mix 1ul plasmids with 100ng mRNA of transposase (Kawakami et al., 2000) and inject it into single-cell stage WT zygotes.
• The embryos were raised to adult and the transgenic founder was identified by direct observation under a fluorescent microscope.