Cell Signaling · Unit 8: Evolution, disease and tools

Hacking the Cell: Reporters, Actuators and Genetic Tools

How do you watch a signal inside a living cell, and how do you start one in a cell of your choosing? This lesson follows green fluorescent protein from a jellyfish to the reporters built from it, explains light-gated channels and other actuators, and shows how transgenes, gene targeting and the Cre and lox system put these tools into one cell type in a mouse. Afterwards you can design a mouse that reports and controls the activity of a chosen neuron type, and say how microscopes got past the limit set by the wavelength of light.

Watch first Tools Behind a Stop, Chosen by Cre · 2:29

A gene copied out of a jellyfish and put into gut bacteria in 1992 made the bacteria glow green, with nothing else from the jellyfish. A cell that builds its own light can be watched while it is alive. The tools that followed let you see a signal as it happens or start one on command, and a set of genetic methods puts either kind into one cell type in a mouse.

The idea: watch a signal, or push one

Two words sort the tools. A reporter is an engineered protein that changes its light when a signal happens, so you can watch it in a living cell. An actuator is an engineered protein that starts a signal on command, usually with a flash of light. Using light and a gene to control cells is called optogenetics, and the word is often stretched to cover reporters used alongside it.

Think of a voltmeter and a switch. A reporter can show that calcium rises in a neuron each time a mouse turns left. Only an actuator can tell you whether firing that neuron is enough to turn the mouse.

From 1961 Osamu Shimomura and Frank Johnson extracted and purified aequorin, a jellyfish protein that gives off blue light when calcium binds it. Traces of a second protein, green and fluorescent, came along in the extract. In 1971 Morin and Hastings renamed it green fluorescent protein, GFP.

“How does fluorescence work?” A molecule absorbs a photon, which lifts an electron to a higher energy level. A little of that energy is lost as heat before the electron falls back and emits a photon. So the light that comes out has a longer wavelength than the light that went in: blue in, green out.

Until 1992 nobody knew whether the GFP gene alone would be enough. The common guess was that a jellyfish enzyme had to finish the protein. That year Douglas Prasher published the cloned gene, and in Martin Chalfie’s laboratory at Columbia the rotation student Ghia Euskirchen put its coding stretch into E. coli. The bacteria fluoresced green.

The structure explains why. GFP is a barrel with a helix through its middle. Once the protein folds, three neighboring amino acids on that helix (serine 65, tyrosine 66 and glycine 67) react with one another, lose water, and are oxidized by oxygen from the air. That leaves a rigid ring system that absorbs violet and blue light and emits green. The recipe is the sequence itself, so many kinds of cell with the gene and oxygen make their own lamp, though the first version folded poorly in warm cells, and a gene joined to it gives a protein with the lamp attached.

Building a reporter: a glowing protein joined to a detector

“What would a synaptic protein look like?” Fuse GFP to gephyrin, a scaffold protein found under inhibitory synapses, and a neuron’s dendrites show rows of bright dots. That is the simplest reporter: it tells you where a protein is.

To make the light depend on a signal, join GFP to a detector you have already met.

  • A lipid reporter. A PH domain that binds PIP3 (see the lesson on lipid anchors and addresses), fused to GFP, moves from the cytosol to the membrane when PI 3-kinase makes PIP3.
  • A pH reporter. A GFP that dims in acid, placed inside a synaptic vesicle (pH about 5.5), brightens when the vesicle fuses and it meets fluid at pH 7.4.
  • A calcium reporter. In the GCaMP family, GFP’s original ends are joined and new ends are cut beside the light-emitting group. Calmodulin (from the calcium lesson) is attached to one new end and a peptide that it grips to the other. When calcium rises, calmodulin wraps around the peptide and plugs the gap in the barrel, which shuts water out from the light-emitting group, and the GFP brightens.
  • A kinase reporter. Put a peptide that the kinase phosphorylates, and a domain that binds the phosphorylated peptide (an SH2 domain, for a tyrosine kinase), between a cyan and a yellow fluorescent protein. Phosphorylation folds the chain. Two fluorescent proteins within a few nanometers pass energy from one to the other without a photon (FRET), so the balance of cyan and yellow light changes as the kinase works.

A reporter also takes part in what it reports. A calcium reporter soaks up some calcium and lets go slowly, so a fast train of spikes can blur into one slow rise.

An actuator: light in, current out

Watch Light In, Current Out · 1:41

In the lesson on the senses and cGMP you met rhodopsin: seven helices holding retinal, which changes shape when it absorbs a photon and sets off a G protein. Some microbes have opsins on a similar plan that skip the relay. Channelrhodopsin-2, from a green alga, is itself an ion channel. Blue light changes its retinal, the pore opens within milliseconds, and cations flow into the neuron. That depolarizes the membrane, and at threshold the neuron fires.

Georg Nagel, Peter Hegemann, Ernst Bamberg and colleagues had shown in 2002 and 2003 that channelrhodopsins from the green alga Chlamydomonas are light-gated channels, and that channelrhodopsin-2 passes cations and works in mammalian cells. In 2005 Edward Boyden, Feng Zhang, Ernst Bamberg, Georg Nagel and Karl Deisseroth reported that hippocampal neurons in culture, given the channelrhodopsin-2 gene, fired action potentials in step with millisecond flashes of blue light. Other laboratories reported similar results at about the same time.

Opsins are sorted by what light does through them:

  • Channelrhodopsins: blue light opens a channel for cations, with little choice among them. The neuron switches on.
  • Halorhodopsins: yellow light drives chloride into the cell. The neuron switches off.
  • OptoXRs: green light activates a rhodopsin whose inner loops come from another G-protein-coupled receptor, so light starts that receptor’s pathway.

A virus can carry the gene into one brain region, though it has room for only a short promoter, and light arrives through an optical fiber.

A drug can do the job of light. In chemogenetics the cell gets an engineered G-protein-coupled receptor that ignores its natural ligand and responds to a synthetic drug, so only cells with the receptor react. A channel from the lesson on ion channels works too: put the TRPV1 gene into one neuron type of a mouse whose own TRPV1 gene has been knocked out, and capsaicin opens a cation channel in those neurons alone (Güler and colleagues, 2012).

How can we make a green animal?

Start with a transgene: DNA built from a promoter (the stretch that sets where and when a gene is read) and a coding sequence. Injected into a fertilized mouse egg, it sometimes joins a chromosome at a random place and is inherited from then on. In 1982 Palmiter and colleagues did this with a metallothionein promoter joined to a growth hormone gene, and the mice grew much larger than their littermates. Use a promoter active in every cell and the coding sequence for GFP, and you have a green mouse.

To change one chosen gene you work in embryonic stem cells, cells from an early embryo that can still become any tissue. Give them DNA that matches the target gene on both sides of your change, and in a few cells the matching stretches swap your version into the gene’s own place. Destroying the gene this way is a knock-out; writing something new into it, such as GFP, is a knock-in. Drugs select the rare correct cells (ganciclovir kills cells that took the DNA up at a random site). The chosen cells, from a strain with a brown “agouti” coat, are injected into an early embryo of a black strain. The pup is a chimera, a patchwork of both, and if the stem cells formed its sperm or eggs, its agouti offspring can carry the change.

“How do you get expression in 2 different neuron types?” The tool is Cre and lox. Cre is an enzyme from a bacterial virus that cuts and rejoins DNA at a 34-base-pair site called loxP. Put two loxP sites around part of a gene, pointing the same way, and Cre removes what lies between them. Make Cre from a promoter that is active in one cell type, and the gene is lost there and nowhere else. To switch a gene on, put a stop signal between two loxP sites in front of it. Where Cre is made the stop is cut out, and the gene is read from then on in that cell and all its descendants, which is how cell lineages are traced. With several fluorescent proteins for Cre to pick among at random, neighboring cells take different colors. For the second neuron type, add a second enzyme of the same kind with its own sites (Flp, from yeast): Cre switches on one gene in one type and Flp another gene in the other.

CRISPR shortens the path: an RNA guide brings a DNA-cutting enzyme to a matching sequence in a fertilized egg, so many knock-outs and knock-ins no longer need stem cells or chimeras.

Design problem

“How can I make a mouse that does this?” First, how can we make a green animal? Then go one step further: design a mouse in which only one type of neuron glows when its calcium rises, and in which you can switch that same type of neuron on with light. Say which reporter, which actuator, and how you make sure that both are made in that cell type and nowhere else.

One way to do it

The green animal is a transgene: a promoter active in every cell, joined to the GFP coding sequence and injected into fertilized eggs.

For the second mouse, breed together three parts.

  1. An address. Cre knocked into a gene that only your neuron type reads.
  2. A reporter. A GCaMP gene behind a lox-flanked stop.
  3. An actuator. A channelrhodopsin gene behind its own lox-flanked stop.

Only cells that make Cre remove the stops, so only they make both proteins.

One trap: GCaMP is read with blue light and channelrhodopsin-2 is opened by blue light, so looking would also be pushing. Use a channelrhodopsin variant that opens with red light.

Past the limit of light

A lens cannot focus light to a point. The glow of one molecule spreads into a blur, and two molecules closer than about half a wavelength merge. Ernst Abbe put the limit as a formula in the 1870s:

smallest separable distance ≈ wavelength ÷ (2 × numerical aperture)

Numerical aperture measures how wide a cone of light the lens collects, about 1.4 for a very good lens. For green light the limit is about 200 nanometers; a synaptic vesicle is about 40 across.

One way past it came through Jennifer Lippincott-Schwartz’s laboratory at the NIH. In 2002 her postdoc George Patterson made a GFP that starts dark and switches on after a flash of violet light. In 2005 the physicist Eric Betzig described to her a microscope that needed such a molecule, and she gave him and Harald Hess room in her laboratory. The method is PALM. Switch on a few scattered molecules, so that no two blurs overlap, and find the center of each blur, which can be done far more precisely than the blur is wide. Let those molecules bleach and repeat thousands of times. The stacked positions make a picture with detail down to tens of nanometers. The paper appeared in 2006, as did two related methods, STORM from Xiaowei Zhuang and FPALM from Samuel Hess.

Other methods take other routes: STED (proposed by Stefan Hell in 1994, first shown to work in 1999) switches off fluorescence around the rim of the spot with a ring-shaped second beam, and structured illumination computes finer detail from striped lighting.

How we measure it

Genetically encoded fluorescent reporters

You join the gene for a fluorescent protein to the gene for a detector, such as calmodulin or a PH domain, and the cell makes the reporter itself. A fluorescence microscope then records the reporter's brightness, position or color over time in living cells, which tells you when and where the signal happened.

Optogenetic stimulation

You express a microbial opsin such as channelrhodopsin-2 in chosen cells and deliver light, in a dish from a lamp or laser and in an animal through an optical fiber. Recording the cell's voltage or the animal's behavior during the light tells you whether activity in those cells is enough to cause the effect.

Gene targeting and Cre/lox conditional genetics

You change one chosen gene in embryonic stem cells by homologous recombination, make chimeric mice from those cells, and breed the change into a line. With loxP sites placed around a piece of DNA, Cre recombinase removes that piece only in the cells that make Cre, so a gene can be removed or switched on in one cell type.

Single-molecule localization microscopy (PALM)

You switch on a few photoactivatable fluorescent proteins at a time, find the center of each separate blur, and repeat for thousands of camera frames. The summed positions give an image with detail of tens of nanometers, well below the diffraction limit of about 200 nanometers.

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Green fluorescent protein — a barrel with the light-emitting group inside
Green fluorescent protein — a barrel with the light-emitting group inside ● LIVE · RCSB PDB 1EMA
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Loading live structure 1EMA from RCSB PDB…

Eleven strands make the barrel. The helix running through its center carries the three residues that react to form the light-emitting group, shielded from water by the barrel wall.

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A channelrhodopsin — seven helices, retinal and a path for cations
A channelrhodopsin — seven helices, retinal and a path for cations ● LIVE · RCSB PDB 3UG9
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Loading live structure 3UG9 from RCSB PDB…

Find the retinal held among the seven membrane-crossing helices. The same protein that catches the photon also forms the pore, so no G protein is needed between light and current.

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GCaMP calcium reporter — GFP joined to calmodulin
GCaMP calcium reporter — GFP joined to calmodulin ● LIVE · RCSB PDB 3EVR
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Loading live structure 3EVR from RCSB PDB…

Calmodulin, with its calcium ions, is wrapped around a short peptide and pressed against an opening in the side of the GFP barrel, next to the light-emitting group.

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Cre recombinase on its loxP site — the enzyme that cuts and rejoins DNA
Cre recombinase on its loxP site — the enzyme that cuts and rejoins DNA ● LIVE · RCSB PDB 1CRX
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Loading live structure 1CRX from RCSB PDB…

Each Cre molecule clamps around half of a loxP site. In the working assembly four Cre molecules hold two loxP sites together so that the DNA strands can be exchanged.

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Osamu Shimomura

Osamu Shimomura 1928–2018

He purified aequorin, a jellyfish protein that emits blue light when calcium binds it, and found beside it the green fluorescent protein that the reporters in this lesson are built from.

In the summer of 1961 Shimomura and Frank Johnson travelled from Princeton to the University of Washington's laboratory at Friday Harbor to learn how the jellyfish Aequorea makes light along the rim of its bell. The accepted idea was that living light came from a fuel, luciferin, and an enzyme, luciferase, and the jellyfish gave them neither. Shimomura set that idea aside and went after whatever the light-emitting substance was. It turned out to be one protein that flashes blue when calcium binds it. From about 10,000 jellyfish he and Johnson purified a few milligrams, and it was named aequorin in a 1962 paper with Johnson and Saiga. A second protein, green and fluorescent, was present in traces in the same extract. They purified that too, and in 1979 he worked out which part of it fluoresces. Aequorin gave physiologists a way to see calcium rise inside a single living cell. The green protein, once its gene was cloned, became a lamp that any cell can be made to build for itself.

✓ verbatim Shimomura, Nobel Lecture, 8 December 2008; Shimomura, Johnson & Saiga, J. Cell. Comp. Physiol. 59:223 (1962).
Douglas Prasher

Douglas Prasher

He cloned and sequenced the gene for green fluorescent protein and gave the DNA to the two laboratories that turned it into a tool.

Prasher cloned the gene for green fluorescent protein at the Woods Hole Oceanographic Institution, on an American Cancer Society grant that began in 1988, and published its sequence in February 1992. By then his funding had run out and he was preparing to leave the work. That year two laboratories, Roger Tsien's in San Diego and Martin Chalfie's at Columbia, each found his paper and telephoned him. He gave the DNA to both. Tsien's Nobel lecture records that Prasher's one condition was a place as co-author if they succeeded, and he is an author of Chalfie's 1994 paper in Science and of the 1994 paper in which Roger Heim and Tsien reported GFP variants with altered colors. Prasher left academic science and was not among the three who shared the 2008 Nobel Prize in Chemistry. In his banquet speech Tsien named him as an example of the many people whose contributions synergized with the laureates' own. From 2012 Prasher worked in Tsien's laboratory. His clone is what let any laboratory make GFP without a jellyfish.

✓ verbatim Prasher et al., Gene 111:229 (1992); Chalfie, Nobel Lecture, 8 December 2008; Tsien, Nobel Lecture, 8 December 2008; Tsien, Nobel banquet speech, 10 December 2008.
Martin Chalfie

Martin Chalfie 1947–

His laboratory showed that the GFP gene by itself makes a bacterium, and then the neurons of a living worm, fluoresce green, so GFP could mark gene expression in a living animal.

Chalfie studied the touch-sensing neurons of the nematode C. elegans, a transparent worm about a millimeter long. His markers for those neurons required killing and fixing the animal. In April 1989 he heard a seminar that described a green fluorescent protein from a jellyfish, and saw that in a transparent worm such a protein could mark living cells. The doubt was whether the gene would be enough, since the common view was that a jellyfish enzyme was needed to make the protein fluoresce. In September 1992 Douglas Prasher sent him the cloned DNA. Chalfie chose to copy out only the protein-coding stretch by PCR, and the rotation student Ghia Euskirchen put it into E. coli. The bacteria fluoresced green on 13 October 1992. Yuan Tu, a technician in the laboratory, then expressed the gene in worm neurons, and the paper appeared in Science in February 1994. Chalfie shared the 2008 Nobel Prize in Chemistry with Shimomura and Roger Tsien. After that paper, a protein's place in a living cell could be read by fusing its gene to the gene for GFP.

✓ verbatim Chalfie, Nobel Lecture, 8 December 2008; Chalfie, Tu, Euskirchen, Ward & Prasher, Science 263:802 (1994).
Jennifer Lippincott-Schwartz

Jennifer Lippincott-Schwartz 1952–

Her laboratory used GFP to measure how proteins move in the membranes of living cells, made a GFP that light can switch on, and hosted the work on the PALM microscope.

In the mid-1990s Nelson Cole of Lippincott-Schwartz's laboratory tagged a Golgi protein with GFP and watched it in living cells at the NIH. They bleached one small spot with a laser, and unbleached molecules moved in and refilled it. The method, called FRAP, measures how fast a protein moves, and their 1996 paper reported that proteins thought to be anchored in the Golgi diffuse rapidly and freely within its membranes. In 2002 her postdoc George Patterson engineered a GFP that starts dark and switches on with a flash of violet light. In 2005 the physicist Eric Betzig, who then had no laboratory, described to her a way around the resolution limit of the light microscope that needed such a protein. She gave Betzig and Harald Hess space in her laboratory and helped find money for parts. The PALM paper appeared in 2006 with nine authors, and Betzig shared the 2014 Nobel Prize in Chemistry. The method places single proteins inside a cell to within tens of nanometers.

✓ verbatim Betzig et al., Science 313:1642 (2006); Patterson & Lippincott-Schwartz, Science 297:1873 (2002); Cole et al., Science 273:797 (1996); Betzig, Nobel Lecture, 8 December 2014.
Test yourself
Question 1 of 8 Score 0 / 0

Which statement correctly separates a reporter from an actuator?
Sources & Integrity

Every structure, slide, simulation, and clip on this page is listed below with the source it came from. None of the 3D structures is drawn by hand or by AI: each one loads by accession ID from RCSB PDB or PubChem. This panel is generated from the lesson's own data file, so what it lists is what the page loads.

Molecular structures live data

  • Green fluorescent protein — a barrel with the light-emitting group inside — RCSB PDB 1EMA · coordinates CC0 · GFP from Aequorea victoria (S65T variant), Ormö et al., Science 1996
  • A channelrhodopsin — seven helices, retinal and a path for cations — RCSB PDB 3UG9 · coordinates CC0 · Chimera of channelrhodopsin-1 and -2 in the closed state, Kato et al., Nature 2012
  • GCaMP calcium reporter — GFP joined to calmodulin — RCSB PDB 3EVR · coordinates CC0 · GCaMP2 with calcium bound, Wang et al., Structure 2008
  • Cre recombinase on its loxP site — the enzyme that cuts and rejoins DNA — RCSB PDB 1CRX · coordinates CC0 · Cre bound to loxP DNA, Guo, Gopaul & Van Duyne, Nature 1997

Scientist profiles & quotes

  • Osamu Shimomura — Shimomura, Nobel Lecture, 8 December 2008; Shimomura, Johnson & Saiga, J. Cell. Comp. Physiol. 59:223 (1962).
  • Douglas Prasher — Prasher et al., Gene 111:229 (1992); Chalfie, Nobel Lecture, 8 December 2008; Tsien, Nobel Lecture, 8 December 2008; Tsien, Nobel banquet speech, 10 December 2008.
  • Martin Chalfie — Chalfie, Nobel Lecture, 8 December 2008; Chalfie, Tu, Euskirchen, Ward & Prasher, Science 263:802 (1994).
  • Jennifer Lippincott-Schwartz — Betzig et al., Science 313:1642 (2006); Patterson & Lippincott-Schwartz, Science 297:1873 (2002); Cole et al., Science 273:797 (1996); Betzig, Nobel Lecture, 8 December 2014.

Quotes are verbatim from the cited source. Profile text is original to this site and was checked against the sources cited.

Animated explainer

Tools Behind a Stop, Chosen by Cre · narration: an AI narrator voice (Gemini, Charon) reading narration written from this lesson's text · animation: AI-generated in code (Manim) by Claude, a schematic with no drawn molecular structures, plots computed from their equations · captions from the narration script.

Section films

  • Light In, Current Out · narration: an AI narrator voice (Gemini, Charon) reading narration written from this lesson's text · animation AI-generated in code (Manim) by Claude, a schematic with no drawn molecular structures · captions from the narration script.

Images & figures

This lesson carries no raster figures. Every structure is rendered live from RCSB PDB or PubChem, and every plot is drawn from its equation in the browser.

integrity Machines check the mechanics. The build confirms that every local file exists, that every question's answer key points at a real choice, and that any AI-generated scientific figure names the person who checked it; a weekly job re-resolves every accession ID against its database. Questions are original; MCAT-style items are written for this site and are not real AAMC questions. Banned sources (the Tymoczko textbook, publisher decks, copyrighted figures) are not used.

Like I'm five

Some jellyfish carry a tiny part that glows green. Scientists copied the instructions for that part into the cells of mice, so that chosen cells light up, and light up more when they are busy. They also borrowed a part from pond algae that works like a light switch: shine blue light on a cell that has it, and the cell turns on. So now they can watch a cell work, and they can also tell it when to work.
High school

Cells send signals with molecules that are too small and too fast to see. Two kinds of engineered protein get around that. A reporter is built from green fluorescent protein, a jellyfish protein that glows green under blue light, joined to a part that senses something such as calcium, so the cell glows brighter when calcium rises. An actuator works the other way round: channelrhodopsin, from an alga, is a pore that opens when blue light hits it, so a flash of light makes a nerve cell fire. Both are proteins, so a gene is all a cell needs to make them, and genetic methods such as the Cre and lox system place that gene in one chosen kind of cell in a mouse.
College

A reporter is a genetically encoded biosensor: a fluorescent protein fused to a detector domain so that its brightness, position or color changes with a signal. GFP suits the job because its chromophore forms from three of its own residues and needs only oxygen, so the gene is enough in many kinds of cell. GCaMP joins a circularly permuted GFP to calmodulin and a calmodulin-binding peptide and brightens when calcium binds. Kinase reporters put a substrate peptide and a phospho-binding domain between two fluorescent proteins and report by FRET. An actuator starts a signal: channelrhodopsin-2 is a seven-helix, retinal-binding protein that is itself a cation channel opened by blue light, and halorhodopsin moves chloride inward to silence a cell. Chemogenetic receptors do a similar job with a drug. Targeting to a cell type comes from genetics: transgenes with chosen promoters, knock-ins made by homologous recombination in embryonic stem cells, and Cre recombinase, which removes DNA between loxP sites only in cells that make Cre. Localization microscopy such as PALM then places single switched-on molecules to within tens of nanometers, below the diffraction limit of about 200 nanometers.
Graduate

Each tool here trades one property for another, and choosing well means knowing the numbers. GCaMP-type reporters bind calcium with affinities of a few hundred nanomolar and with cooperativity, so their response is steep and saturates. They release calcium over hundreds of milliseconds, so they low-pass filter a spike train, and at the concentrations needed for imaging they add calcium buffering of their own. FRET reporters for kinases report the balance between the kinase and the phosphatases that act on the reporter's substrate peptide, and their ratio changes are often small. Channelrhodopsin-2 has a small single-channel conductance compared with most neuronal channels, so a neuron must make a great many copies, and it closes about ten milliseconds after the light ends, which limits how fast a spike train it can drive. Its pore also passes protons and some calcium. In PALM the localization precision scales roughly as the width of the blur divided by the square root of the photons collected, so a fluorescent protein that yields a few hundred photons is placed to within about 2 to 25 nanometers, as in the first PALM paper, at the cost of thousands of camera frames and so of time resolution. The field is pushing these tools toward non-excitable cells, protein-protein interactions, small GTPases, and control of where in a cell a protein sits. The open question underneath is one of specificity. An opsin controls one variable, membrane voltage, cleanly. Can an actuator be built that switches on one kinase or one GTPase, at one place in a cell, with the natural size and time course of the signal, and can a reporter confirm that without changing the signal it reads?
A flash in the sink

Shimomura needed a way to switch the light of his jellyfish extract off and on, so that he could purify the substance before it used itself up. He found that the extract went dark in acid at pH 4 and glowed again when he neutralized it. Then he poured a dark extract into a sink and the sink lit up bright blue. Seawater from an aquarium was running into that sink. The ingredients of seawater are known, and he soon had the one that mattered: calcium. A protein that gives light when calcium arrives is a calcium reporter, and physiologists later injected aequorin into cells for that purpose.

— Shimomura, Nobel Lecture, 8 December 2008.
The microscope was borrowed

The glowing bacteria were not seen in Martin Chalfie's own laboratory, which had no working fluorescence microscope. Ghia Euskirchen, a rotation student who had just finished a master's degree on fluorescence in Columbia's engineering school, took the culture to a microscope there. She recorded the green cells in her notebook on a page headed Tuesday 13 October. Chalfie reproduced that page in his Nobel lecture and noted under it that the microscope she used was not in his laboratory. By his account the cloning choice mattered too: groups that moved the whole cloned fragment, with extra non-coding jellyfish DNA around the gene, saw no fluorescence.

— Chalfie, Nobel Lecture, 8 December 2008 (the notebook page of Ghia Euskirchen, 13 October 1992).
The giant mice of 1982

The answer to "How can we make a green animal?" begins with mice that were not green but big. In 1982 Richard Palmiter, Ralph Brinster and colleagues joined the promoter of a mouse metallothionein gene to a rat growth hormone gene and injected the DNA into fertilized mouse eggs. Some of the mice that developed carried the fusion gene, made growth hormone under the control of the borrowed promoter, and grew much larger than their littermates. The experiment holds the whole logic of a transgene: the promoter decides where and when, and the coding sequence decides what is made.

— Deppmann, signaling lecture 16 (Hacking); Palmiter et al., Nature 300:611 (1982).

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