Papp A et al. (1991) Worm Breeder's Gazette "Improved Injection Setup!"

Papp A, Mancillas JR

[Worm Breeder's Gazette, 1991]

We have devised a very convenient, semi-automated injection setup closely based on the system of Mello and Ambros (wbg 11(4):7). It provides many of the advantages of the digital automated systems available, at a minute fraction of the cost. Our system is very similar to that described by Mello and Ambros, and anyone with a system like theirs can upgrade. The main difference is that the 3-way ball valve, used to control the duration of injection flow, has been replaced with an electronic solenoid-operated valve. This permits foot pedal (or any other way you'd prefer to operate an electric switch) control, and leaves both hands free to deal with micro- manipulation and focusing. In addition, the solenoid operated valve can open and close in a fraction of a second, permitting very precise control of flow. If the needle happens to be in the wrong place, a quick tap will show this without appreciably damaging the worm. at high pressure, quick on/off pulses also appear to aid in unclogging clogged needles, while at low pressure the system is ideal for pulse- filling oocyte nuclei or gonads. By varying the pulse time, it is possible to compensate for the flow characteristics of individual needles. By selecting the right match of components, it is possible to construct a system as drawn below which is extremely compact, attaching directly to the pressure source, requiring no cut hypodermic needles or tubing (except the piece connected to your needle) and occupying no bench space at all. Shown not to scale below, the entire system that attaches to a pressure regulator measures only 3' x 5' x 3/4', and minimizes dead gas space. Fittings such as those described by mello and ambros can be used; however, the key is to replace the 3- way ball valve (b41x52) with a solenoid valve such as the nupro 110vac model. All metal-to-metal connections should be wrapped in teflon-based tape. Controlled pressure can be supplied to this system with a nitrogen tank via a high-pressure regulator or with a pump or in-house compressed air via a low-pressure regulator. For a complete parts list and any additional information, please contact us. In addition, a pre-assembled and tested complete system including foot pedal controller and the tubing that attaches directly to your needles can be obtained from tritech research (see above address and phone number). As an introductory offer to worm breeders, the system is available for $210, or $360 with high-quality pressure regulator and tank fitting (approximately the retail cost of the unassembled components). [See Figure 1]

Beitel GJ et al. (1990) Worm Breeder's Gazette "Rescue of the lin-9 Synthetic Multivulva Phenotype by Germline"

Horvitz HR, Beitel GJ

[Worm Breeder's Gazette, 1990]

A multivulva phenotype can require the presence of mutations in two separate genes, despite the fact that each mutation results in an apparently wild-type phenotype by itself (Ferguson and Horvitz 123:109121, 1989). Mutations that can cause this synthetic multivulva (syn Muv) phenotype have been grouped into two classes, class A and class B, such that a double mutant carrying one mutation in each class will have the syn Muv phenotype. lin-9(n112) III is a class B mutation that results in an apparently wild-type phenotype by itself. lin-9 maps 0.01 m.u. to the left of unc-32 III, which has been rescued in germline transformation experiments by John Sulston (Hope et al., WBG 10(2) p. 97). Using the gonadal syncytial injection method developed by Craig Mello et al. (WBG 11(1) p. 18), we microinjected cosmids from the unc-32 region along with a dominant rol- 6 marker into lin-9(n111); lin-15(n433) animals, which are temperature- sensitive for the Muv phenotype and had been raised at the permissive temperature. A ts strain was used to generate animals sufficiently healthy for efficient microinjection, as well as to potentially minimize the amount of rescue activity required from the injected cosmid. The lin-15 allele used, n433, is a class A syn Muv mutation that results in a wild-type phenotype in the absence of a class B syn Muv mutation. The major approach we have used to test cosmids near unc-32 is to raise the F1 progeny of the injected animals at the permissive temperature, clone F1 rollers, raise their progeny at the restrictive temperature and then score the F2 progeny for rescue of the Muv phenotype and the presence of rollers. We chose this approach based on observations that some genes show only F2 rescue in gonadal syncytial injection experiments (Craig Mello and Gian Garriga, personal communications). The cosmid ZK637, which rescues unc-32, also rescues lin-9. Using ZK637, 14 out of 24 lines that transmit rollers show rescue of the Muv phenotype. Interestingly, both the rol-6 and lin-9 rescue activities seem to be incompletely penetrant, because animals from rescued lines with any of the phenotypes Rol non-Muv, Rol Muv, non-Rol Muv or non- Rol non-Muv can produce progeny with the Rol non-Muv rescued phenotype. A similar phenomenon has been previously observed by Craig Mello for rol-6 and several other genes (see article in this issue). In preliminary experiments testing for F1 rescue by injecting ZK637 and raising the F1 progeny at the restrictive temperature, approximately 10% of the F1 rollers have been non-Muv. However, Muv Rol animals ruptured at the vulva less frequently and survived to adulthood more often than did Muv Rol animals generated with just rol- 6 DNA, suggesting that partial rescue might have occurred. In several cases, cloned F1 non-Muv non-Rol animals subsequently produced Rol non- Muv animals in the F2. The only overlapping cosmid left of ZK637 is K01F9. Injection of K01F9 has not rescued lin-9 in seven lines of rollers. We are attempting to localize lin-9 on ZK637 both by testing other overlapping cosmids and by subcloning ZK637.

Barbazuk BW et al. (1995) Worm Breeder's Gazette "Correlation of the Genetic and Physical Maps Within the rol-3 Region of LGV (left)."

Baillie DL, Barbazuk BW

[Worm Breeder's Gazette, 1995]

Correlation of the Genetic and Phvsical maps within the rol-3 region of LGV (left) W. Bradley Barbazuk and David L. Baillie Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC. CANADA, VSA 1 S6 In an attempt to identify the physical location of the rol-3 (V) gene we have begun the systematic germ-line transformation rescue of lethal mutations within zones 16-18 of LGV (JOHNSEN and BAILLIE, GENETICS 129: 735-?52). Using H. Kagawa's placement of unc-68 (WBG 12(4)) as a starting point we began to inject cosmids to the right of C46C6 into N2 hermaphrodites. In doing so we were able to obtain a battery of strains each carrying cosmids singly, or multiply, in stable arrays which were then used as mini-duplications in subsequent crosses to lethal bearing strains. In total we used eight cosmid bearing strains and one YAC bearing strain in the rescue of 13 lethal mutations including rol-3 (Table 1). Our germ-line transformation rescue data has enabled us to split up and order some of the lethal mutations within the zone 16-18 gene clusters. (Figure 1). We would like to thank C. Mello and J. Priess for providing a strain carrying an array composed of ZK307, F25G6 and FOlH8, as well as providing purified DNA for many of the cosmids injected.

Mitani S (1995) Worm Breeder's Gazette "Integration of extrachromosomal DNA arrays into a chromosome by UV- irradiation"

Mitani S

[Worm Breeder's Gazette, 1995]

To work with transgenic C. elegans animals, it is common to construct strains with extrachromosomal DNA arrays by microinjection of DNA (Mello et al., 1991). These strains, however, are mosaic in terms of the transgene and sometimes need a second step to integrate the transgene into a chromosome. The most common method to do this is to irradiate the parent animals with gamma ray from a l37Cs source. Because UV light induces the nematodes similar gene rearrangements with those by gamma ray irradiation (Stewart et al., 1991), I developed a UV irradiation method which may substitute a common gamma ray method for integration of DNA into a chromosome of C. elegans. Parent strains were irradiated with a UV light (254 nm) using a UV cross linker (commonly used to fix polynucleotides to nylon membranes; spectrolinker, Spectronics) at a dose of 300 J/m2 (the optimal dose among examined [ranging from 150-1000 J/m2]). Irradiated parents were allowed to lay eggs and healthy Fl animals were picked onto individual plates. F2 rollers from each plate were singled and strains which produced only roller animals were identified later, as the gamma ray method. I have found the probability of integration is high enough for routine experiments: about 5%. Since UV light is much easier to access for most researchers than the gamma ray source, it appears a convenient alternative to obtain stable transgenic animals with reporter gene fusion or heat-shock expression.

Vatcher GP et al. (1997) Worm Breeder's Gazette "SuperCosmid Strikes Again"

Vatcher GP, Kuervers LM, O'Neil NJ, Baillie DL

[Worm Breeder's Gazette, 1997]

In recent articles, WBG 14(2), and this issue, we reported on the use of a cosmid transgenic library to correlate the physical and genetic maps. Using these transgenic strains we have rescued the mutant phenotypes of several lethal genes on LGIII left in the region defined by the overlap of sDf125 and sDf127. The phenotypes of mel-32, let-721, and let-725, F2 egg lethal, were completely rescued by cosmid C05D11 but not by the flanking cosmids C16A3 or T26A5. C05D11 also completely rescued the lethal phenotypes of let-713 (arrests as sterile adults), and let- 756 (an early larval lethal). C05D11 has been affectionately termed SuperCosmid, based on the fact that it rescued 5! essential genes from our collection. All rescued strains were tested for the presence of cosmid with PCR using a vector specific primer set. Outcrossing to wild type and recovery of the lethal or maternal effect lethal phenotype also confirmed a valid rescue. C05D11, a 50kb cosmid, has been subcloned into many smaller fragments which have been injected into the syncitial gonad of C. elegans to produce new transgenic strains. In collaboration with Danielle Thierry-Mieg and Regine Roubin, WBG 14 (4), let-756 was determined to be ORF 4, a heparin-binding growth factor gene (a fibroblast growth factor homolog). The rescue of let-721 with overlapping subclones has shown that let-721 corresponds to ORF 12. This gene has a very high homology with a human cDNA of unknown function. mel-32 has been rescued with a subclone containing only ORF 11 which is the only known C. elegans homolog of the ubiquitous serine hydroxymethyltransferase. In collaboration with Heinke and Ralf Schnabel we are further characterizing this gene. Rescue experiments have narrowed the position of let-713 down to two ORFs and let-725 down to an operon containing three ORFs. Future sequence analysis of these mutant genes will allow the correlation of changes in the DNA sequences (and the subsequent protein sequences) with their lethal phenotypes. Lethal and maternal effect lethal genes are essential for survival; therefore, their assignment to specific ORFs will help define which genes are required for normal growth and development.

Nishiwaki K et al. (1991) Worm Breeder's Gazette "Molecular cloning of the emb-5 gene"

Miwa J, Nishiwaki K, Sano T

[Worm Breeder's Gazette, 1991]

Temperature-sensitive maternal effect mutations in the emb-5 gene (1) show premature second E cell division and abnormal gastrulation at nonpermissive temperatures. Embryos arrest as balls of several hundred differentiated cells around the lima bean stage (2). We previously reported identification of a Bergerac Tc1 closely linked to emb-5 (WBG 11, 1, 39). We obtained several overlapping cosmids (from J. Sulston and A. Coulson) closely linked to this Tc1 containing region so that some of the cosmids possibly contain the emb-5 gene. We injected 4 overlapping cosmids individually into emb-5(hc61) according to the method of C. Mello and V. Ambros (personal communication). Of these, two overlapping cosmids, C56E12 and C17H4, were found to have transforming activity, indicating that the activity was within a 20kb overlapping region. To limit the region harboring the transforming activity, we examined transformation after cutting C56E12 with various restriction enzymes, as described by S. Kim et al. (3), and found that region within the 10.3kb fragment. Germline transformants grew slowly at nonpermissive temperature and revealed a wide range of E cell phenotypes from mutant type to completely rescued type similar to wild type. Using an internal 1.7kb SalI fragment ( SalI digestion resulted in no rescue) as a probe, we detected a single 5.4kb mRNA in both wild type and hc61. We obtained several cDNA clones from B. Barstead's cDNA library with the same probe. One of the clones had an insert of about 3.6 kb cDNA and the sequence of this cDNA revealed the 1121 aa C-terminal portion of the putative gene product. No significant homology was found when this amino acid sequence was compared with those for other proteins in the SwissProt and the NBRF databases. Preliminary results showed amino acid change from Ala to Glu at 663rd aa from C-terminus in hc61. Currently, we are attempting lacZ fusion experiments using the vectors provided by A. Fire (4) to understand the localization of the gene product.

Fitzgerald K et al. (1992) Worm Breeder's Gazette "Structure/Function Analysis of the lin-12 Protein"

Fitzgerald K, Greenwald IS

[Worm Breeder's Gazette, 1992]

We are currently trying to establish the relationship of various lin -12 structural motifs to their function in vitro. As a first step in this process we have attempted to map functional domains of the lin -12 protein using lin 12/glp-1 protein chimeras under lin-12 regulation. Two chimeras were constructed (see the figure below) by fusing corresponding introns or exons of lin-12 and glp-1 (Yochem and Greenwald Cell 58: 565, 1989; Austin and Kimble Cell 58: 565, 1989). Each construct contains 3.4 kb of lin-12 5' flanking sequence, which is sufficient to allow a lin-12 genomic DNA clone to complement a lin-12 null mutation (Wilkinson and Greenwald WBG 12: 61, 1992). Each construct was injected at a concentration of 6ug/ml along with a dominant rol-6 marker (Mello et al, EMBO J 10: 3959, 1991) at lOOug/ml into N2 wild type hermaphrodites. Arrays from the resulting Roller lines were then crossed into a lin-12 (n676n909am)background and assayed for rescue of the Lin-12 (0)sterility and protruding vulva defects. In each case the chimera containing array was able to complement lin 12(n676 n909 ).These results indicate that lin-12 /glp-1 chimeras most likely will not be useful for mapping functional domains of lin-12 (or glp-1 ).However, these results are interesting in that they strongly support the hypothesis of Lambie and Kimble (Development 112: 231, 1991), who suggested that lin-12 and glp-1 are functionally interchangeable based on the novel "Lag" phenotype of a lin-12 (0)glp-1 (0) double mutant. (Additional support for this hypothesis was also provided by Mango et al, Nature 352: 811, 1991.) We have now begun a structure/function analysis of the lin-12 protein by making specific deletions and point mutations, starting with deletions of various EGF like repeats in the extracellular portion of the protein.

Dunn MA et al. (1999) Worm Breeder's Gazette "A PIE-1-BASED VECTOR FOR MATERNAL EXPRESSION OF GFP FUSIONS."

Reese KJ, Seydoux GC, Dunn MA

[Worm Breeder's Gazette, 1999]

We have used sequences from the pie-1 gene to construct a vector designed to express GFP fusions in the adult germline and in early embryos. The pie-1 gene encodes a maternal RNA transcribed in the adult germline and inherited maternally in embryos (Mello et al., 1992, 1996; Tenenhaus et al., 1998). We have shown previously that an 8kb genomic fragment from the pie-1 locus is sufficient to tag PIE-1 with GFP and rescue a pie-1 mutation (Fig. 1; Dunn et al., Reese et al., 1998 East Coast Worm Meeting Abstracts). We now have modified this construct to allow expression of heterologous GFP fusions. The resulting vector (Fig. 2) contains the pie-1 promoter, the pie-1 3UTR, and the pie-1 large intron, all of which appear to be important for robust expression in the adult germline and in early embryos. We have tested this new vector in 4 configurations: as is (GFP alone), and with one of three different open-reading frames inserted at the SpeI site after the GFP: his-11 (histone H2B), C36E8.5 (beta tubulin), and act-1 (actin). These constructs were injected into N2 hermaphrodites using the Fire labs complex array injection method which permits germline expression of transgenes (Kelly et al., 1997, Genetics 146: 227-238). For all constructs, we obtained a number of lines (10% to 50% of all roller lines) with GFP expression in the germline and in early embryos. In most lines, expression was restricted to the F2 generation after injection. In all cases, the subcellular localization observed matched that expected for each fusion: throughout cells for GFP alone, on DNA for GFP:histone H2B, on centrosomes and meiotic (and more weakly mitotic) spindles for GFP:beta-tubulin, and on cell cortices and cell division remnants for GFP:actin. (In one GFP:actin line, we also saw nuclear GFP). In addition to germline expression, all lines also expressed GFP in a number of somatic cells. We are hopeful that this vector could be used by others to express additional GFP fusions in the germline and in early embryos. Anyone interested can obtain the pie-1 vector from Melanie Dunn at mdunn@jhmi.edu. Fig. 1. Fig. 2.

Tedesco PM et al. (1992) Worm Breeder's Gazette "Integrated Transgenic Lines Rescue Low Fertility in Age Strains"

Johnson TE, Tedesco PM

[Worm Breeder's Gazette, 1992]

Our main focus is on the positional cloning of age-1 .We based our initial cloning strategy on the cosegregation of Age and a reduced fertility (Fer) phenotype in backcrosses of age-1 to N2 ,making the assumption that the same mutational event gave rise to both traits. This assumption simplified the cloning because it is much easier to identify cosmids complementing the Fer trait than the Age trait. We showed that the fer-15 region was responsible for the reduced fertility by deficiency mapping and presented the corresponding physical map (WBG 11.4:14,15). Cosmid T13F9 detected end points of deficiencies immediately to the left and right of fer-15 ;thus T13F9 contains fer-15 ,while age-1 maps closer to unc-4 (WBG 11.4:74). As a final proof that the Age phenotype is separable from Fer we made a series of transgenic animals using the rol-6 coinjection strategy; we also included an actin-4 plasmid (pA4 Gal,Jocelyn Shaw) that was reported to increase the frequency of line formation (Mello, WBG 11.3:12). The injections were successful when we coinjected T13F9 and we saw complementation for fer-15 (b26)at 25 C, although the level of complementation was low. At 20 C little effect on fertility was observed (Table 1). We isolated two independent integrated lines starting with one of the cosmid/Rol-6 arrays and have found that one of these lines complements fertility quite well at 20 C and at 25 C. The other integrated line fails to complement at 20 C and has lower levels of complementation at 25 C. We were not able to get complementation using C26D10 which overlaps extensively with T13F9 and which was reported by Brister and L'Hernault (WBG 11.4:21) to contain fer-15 . These results show that integrated arrays work better for complementation than do extrachromosomal arrays. The quantitative approach we've employed is useful because, unlike qualitative analyses, quantitative analysis can provide better insight into the the level of function of integrated arrays positioned in different chromosomal milieus. We are planning life span assays of these integrated transgenics and hope that they will confirm the fact that the Age and Fer traits are genetically separable. [See Figure 1]

Fire A et al. (1991) Worm Breeder's Gazette "Of rollers and twitchers"

Harrison SW, Fire A

[Worm Breeder's Gazette, 1991]

Craig Mello has in the last few gazettes introduced the use of rol-6 as a coselection marker and some highly efficient and user-friendly techniques for creating transformed lines. We have been using these techniques, and have several observations that should be useful to other transformers. Getting a few good roller lines: Craig's technique of high volume distal injections yields large numbers of F1 animals that express the injected DNA (this is seen both with rol-6 and with lacZ fusions). Cloning of all of these animals is very tedious. To make a set of rol-6 transformed lines from a given construct, we inject 9-12 animals, placing three injected animals each on 3-4 medium size (6cm) plates. After a week the plates are starved, with mostly F2 progeny. From each initial plate three large chunks are placed onto a large seeded plate (9cm). Larval rollers can easily be spotted after a day or so, we generally pick 6-8 for each initial plate, and of these about 90% yield transformed lines. Different F2- F3 clones from the same initial plate need not be independent, so we only analyze one clone from each initial plate, generally the one with the highest transmission frequency. This procedure yields 3-4 independent high transmission lines for each injection set. Twitchers: When mixtures of the unc-22 antisense plasmid pPD10.46 and a second plasmid are injected at high volume into the distal arm, 3-10 twitchers are seen per injected adult. Unlike the roller injections, a relatively high fraction of these animals yield transformed lines: between 40% and 80%. This suggests that pPD10.46 does not cause twitching except when present at high copy number, and thus that twitching is a more reliable indicator of array formation in the F1 than F1 rolling or -gal staining. cis effects: Any marker used for co-transfomation experiments has the potential to affect the expression of co-transformed DNAs. Although these effects do not preclude the use of a marker, they should be taken into account when interpreting results (particularly unexpected results). We find that both pPD10.46 and the rol-6 plasmid pRF4 can have distinct effects on the expression of other constructs in mixed arrays.