Generating Transgenic Worms via Mos1 Transposition

Transgenic worms were made by injection into EG4322 (ttTi5605; unc-119(ed3)) or EG4316/EG5003 (unc-119(ed3) III; cxTi10882 IV) animals 1 (link). The standard injection mix consisted of 50 ng/μl repair template, 50 ng/μl Mos1 transposase (either pJL44(Phsp-16-48::transposase) or pJL43.1(Pglh-2::transposase)), 10 ng/μl pCFJ70 (Pmyo-3::twk-18(cn110)), 5 ng/μl pGH8 (Prab-3::mCherry), 5ng/μl pCFJ104 (Pmyo-3::mCherry) and 2.5 ng/μl pCFJ90 (Pmyo-2::mCherry). In later direct insertion experiments pCFJ70 (Pmyo-3::twk-18(cn110) was omitted from the injection mix. unc-119 animals are severely paralyzed and egg-laying defective. Therefore, L1-L2 animals were manually distributed across a lawn of OP50 and very young adults were selected for injection. Injected animals were individually transferred to standard NGM plates and placed at 15°C. Plates were scored for the number of phenotypically rescued F1 animals 3 days after injection.

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Frøkjær-Jensen C., Davis M.W., Hopkins C.E., Newman B., Thummel J.M., Olesen S.P., Grunnet M, & Jorgensen E.M. (2008). Single copy insertion of transgenes in C. elegans. Nature genetics, 40(11), 1375-1383.

Publication 2008

Animals Mos1 transposase Transgenic Transposase Worms Young adults

Corresponding Organization :

Other organizations : University of Utah, Howard Hughes Medical Institute, University of Copenhagen, Danish National Research Foundation

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Protocol cited in 113 other protocols

Variable analysis

independent variables

Injection into EG4322 (ttTi5605; unc-119(ed3)) or EG4316/EG5003 (unc-119(ed3) III; cxTi10882 IV) animals
Mos1 transposase (either pJL44(Phsp-16-48::transposase) or pJL43.1(Pglh-2::transposase))
PCFJ70 (Pmyo-3::twk-18(cn110))

dependent variables

Number of phenotypically rescued F1 animals 3 days after injection

control variables

Repair template concentration (50 ng/μl)
PGH8 (Prab-3::mCherry) concentration (5 ng/μl)
PCFJ104 (Pmyo-3::mCherry) concentration (5ng/μl)
PCFJ90 (Pmyo-3::mCherry) concentration (2.5 ng/μl)
Temperature (15°C)

controls

Positive control: unc-119 animals (severely paralyzed and egg-laying defective)
Negative control: Not explicitly mentioned

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