Single-copy insertion of transgenes in Caenorhabditis elegans

Abstract

At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.

Figure 1

Schematic presentation of Mos1 mediated Single Copy Insertion (MosSCI). A Mos1 transposon located at a non-coding locus was isolated by the NemaGENETAG consortium. The Mos1 element can be excised by transposase expression resulting in a double-strand break in the chromosome. Presumably the 3′ ends from the left (L) and right (R) flanks invade and anneal to homologous regions in the extrachromosomal array. The break can then be repaired by synthesis-dependent strand annealing. The positive selection marker unc-119(+) and the gene of interest are inserted into the genome by gene conversion. The extrachromosomal array contains a source of transposase (hsp::transposase) and two negative selection markers, twk-18(gf) and fluorescent mCherry markers. twk-18(gf) is a temperature-sensitive dominant mutation in a potassium channel, which paralyzes the animals at 25°C but not at 15°C. mCherry markers are expressed in the pharynx, body muscle, and nervous system for visual identification of array-carrying animals. Following loss of the array, single-copy transgenic animals are isolated. L and R homologous regions = 1.4 kb.

Figure 2

Single-copy insertions of transgenes. (a) Schematic of the targeting construct containing the Punc-122::GFP transgene and unc-119(+) rescue marker (4.3 kb total) flanked by DNA homologous to the ttTi5605mos insertion site. (b) Expression of the Punc-122::GFP transgene in MosSCI insertion strains is restricted to the coelomocytes (arrows). Remaining visible fluorescence is non-specific gut granule fluorescence. Image is of an adult hermaphrodite, anterior is left. DIC above, fluorescence image below. (c) PCR verification of inserted Punc-122::GFP transgenes. The forward primer anneals to the genomic region outside of that contained in the targeting construct and the reverse primer is in the C. briggsae unc-119(+) selectable marker. A PCR band of expected size (1.8kb) from all MosSCI insertion strains (EG4441-EG4450) confirm insertions are at the targeted locus. We interpret the presence of a dim band from the parent strain carrying the extrachromosomal array as evidence for either low levels of somatic gene conversion or PCR bridging. (d) Southern analysis confirms single copy insertion in 60% of the strains. Genomic DNA was digested with EcoRI and hybridized with a GFP-specific probe. Six of ten strains showed a single, specific band of appropriate size verifying single-copy transgene insertions. Three strains were non-fluorescent (indicated by asterisks) and the Southern blot demonstrated that these transgenes contained short deletions and insertions. The molecular changes in EG4441 and EG4448 were further characterized (Supplementary Fig. 3 and Supplementary Fig. 4). Strain EG4449 was visibly more fluorescent and contains two copies of GFP.

Figure 3

Independent unc-18::mCherry transgenes are expressed at near-endogenous levels. (a) Schematic of unc-18::mCherry and unc-119(+) targeting construct (9.0 kb). The unc-18::mCherry transgene contains upstream and downstream regulatory regions extending to adjacent genes. (b) Comparison of unc-18::mCherry inserted by MosSCI and by biolistic bombardment. unc-18::mCherry was inserted by MosSCI (left) and by biolistic bombardment (right). Confocal images were taken with identical settings to compare fluorescence intensity. Expression is specific to the nervous system but brighter in the biolistic strain UZ566. (c) Transgene copy number is uniform in fluorescent MosSCI lines. Southern blot hybridized with mCherry specific probe shows a single-copy insert of predicted size in the three fluorescent MosSCI unc-18::mCherry lines and variable copy number in the two biolistic lines. The MosSCI line UZ557 was non-fluorescent and is marked by an asterisk. (d) Western blot with UNC-18 polyclonal antibody shows uniform, near-endogenous protein expression levels. Whole-worm lysates from the wild type, unc-18(md299) null mutants, MosSCI strains, and biolistic strains were blotted with an UNC-18 antibody. Specific bands of the appropriate sizes corresponding to wild-type UNC-18, from the endogenous locus, and mCherry-tagged UNC-18, from the transgene, were observed in all fluorescent strains. Transgenic strains generated by MosSCI showed uniform, near-endogenous protein levels compared with variable, higher expression from biolistic strains. unc-18::mCherry degradation bands are marked by asterisks.

Figure 4

MosSCI inserts are expressed in the female and male germlines. (a) A Ppie-1::GFP::histone transgene is expressed throughout the female germline. Scale bar = 100 μm. (b) A Pspe-11::mCherry::histone transgene is expressed in hermaphrodite sperm. Left, mCherry expression from Pspe-11::mCherry::histone is specific to hermaphrodite sperm. Right, overlay with DIC image. Most sperm are found in the spermatheca, although a few can also be seen in the gonad (left) and uterus (right). Scale bar = 10 μm. Right inset, mCherry expression is localized to nucleus. 4x magnification.

Figure 5

MosSCI inserts can be generated directly by injection. (a) Schematic of targeting transgene containing unc-119(+) rescue gene and Punc-122::GFP coelomocyte specific expression transgene. Transgenes can be directly inserted in the P0 animals by co-injection with germline specific Pglh-2::transposase and in the absence of twk-18(gf) negative selection marker. (b) Transgene insertion at target site is verified by PCR. A PCR product of the predicted size is obtained in all strains generated by direct injection. (c) A Southern blot confirms single-copy insertion at the target site. Genomic DNA digested with EcoRI was probed with a GFP specific probe and confirms that seven of eight strains contain the predicted single-copy insert. EG4893 contains two GFP fragments and GFP fluorescence was visibly brighter.