Expanding the Genetic Code of an Animal

Genetic code expansion, for the site-specific incorporation of unnatural amino acids into proteins, is currently limited to cultured cells and unicellular organisms. Here we expand the genetic code of a multicellular animal, the nematode <i>Caenorhabditis elegans</i>

Efficient and Rapid <i>C. elegans</i> Transgenesis by Bombardment and Hygromycin B Selection

<div><p>We report a simple, cost-effective, scalable and efficient method for creating transgenic <i>Caenorhabditis elegans</i> that requires minimal hands-on time. The method combines biolistic bombardment with selection for transgenics that bear a hygromycin B resistance gene on agar plates supplemented with hygromycin B, taking advantage of our observation that hygromycin B is sufficient to kill wild-type <i>C. elegans</i> at very low concentrations. Crucially, the method provides substantial improvements in the success of bombardments for isolating transmitting strains, the isolation of multiple independent strains, and the isolation of integrated strains: 100% of bombardments in a large data set yielded transgenics; 10 or more independent strains were isolated from 84% of bombardments, and up to 28 independent strains were isolated from a single bombardment; 82% of bombardments yielded stably transmitting integrated lines with most yielding multiple integrated lines. We anticipate that the selection will be widely adopted for <i>C. elegans</i> transgenesis via bombardment, and that hygromycin B resistance will be adopted as a marker in other approaches for manipulating, introducing or deleting DNA in <i>C. elegans</i>.</p></div

Hygromycin B independent transmission of integrated transgenes.

<p>Worms expressing the hygromycin B resistance gene and mCherry from the ubiquitous P<i>rps-0</i> promoter and GFP from the pan-neuronal P<i>snb-1</i> promoter (P<i>rps-0</i>::<i>HygB,</i> P<i>rps-0</i>::mCherry, P<i>snb-1</i>::<i>GFP</i>, bombardment 64, strain 64.1.1) were grown as described in Figure S3. Worms were washed off the plate and imaged using a confocal microscope as described in the methods section. The strain was grown and propagated in the absence of hygromycin B.</p

Light-Activated Kinases Enable Temporal Dissection of Signaling Networks in Living Cells

We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-determining processes in the pathway

Light-Activated Kinases Enable Temporal Dissection of Signaling Networks in Living Cells

We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-determining processes in the pathway

Light-Activated Kinases Enable Temporal Dissection of Signaling Networks in Living Cells

We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-determining processes in the pathway

Efficiency of biolistic <i>C. elegans</i> transgenesis using hygromycin B selection.

<p>n.d. – not determined.</p

Light-Activated Kinases Enable Temporal Dissection of Signaling Networks in Living Cells

We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-determining processes in the pathway

Hygromycin B selection of transgenic <i>C. elegans</i> created by biolistic bombardment.

<p>Gold beads coated with the DNA mixture of interest, carrying the hygromycin B resistance gene, are bombarded onto worms spread on an agar plate. Bombarded worms are split onto 10–20 selection plates and left to lay eggs for 2–3 days, at which point hygromycin B is added to the plates. After 4–7 days surviving transgenic worms can be picked.</p

Survival of <i>C. elegans</i> in the presence of hygromycin B.

<p>a) 3000 synchronized L1 larvae were transferred to seeded 6 cm NGM plates and immediately treated with hygromycin B. Images of control and treated plates were acquired 40 hours later. b) Survival assay on hygromycin B dilution. 3000 synchronised L1 larvae were plated on seeded 6 cm plates and immediately treated with hygromycin B at the specified final concentrations. The plates were scored for L4 larvae after 40 hours and for adults after 90 hours. The experiment was performed in triplicate, error bars represent standard deviation. c) Selection of transgenic animals on hygromycin B. 10 synchronized L1 larvae carrying a hygromycin B resistance gene and expressing GFP/mCherry were mixed with the indicated number of wild type L1 larvae on a seeded 6 cm plate and immediately treated with hygromycin B (0.3 mg ml⁻¹). The number of adult transgenic (fluorescence-positive) and non-transgenic (fluorescence-negative) animals was scored after 90 hours. The experiment was performed with two independent hygromycin B resistant transgenic strains.</p