Aby wyświetlić tę treść, wymagana jest subskrypcja JoVE. Zaloguj się lub rozpocznij bezpłatny okres próbny.

Summary

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Abstract

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Introduction

Forward genetic screens have been widely used to generate genetic mutants disrupting various biological processes in model organisms such as Caenorhabditis elegans (C. elegans)¹. Analyses of such mutants lead to the discovery of functionally important genes and their signaling pathways^1,2. Traditionally, mutagenesis in C. elegans is achieved using mutagenic chemicals, radiation, or transposons². Chemicals such as ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) are toxic to humans; gamma-ray or ultraviolet (UV) radiation mutagenesis requires special equipment; and transposon-active strains, such as mutator strains³, can cause unnecessary mutations during maintenance. We have developed a simple approach to induce heritable mutations using a genetically-encoded photosensitizer⁴.

Reactive Oxygen Species (ROS) can damage DNA⁵. The mini Singlet Oxygen Generator (miniSOG) is a green fluorescent protein of 106 amino acids that was engineered from the LOV (Light, Oxygen, and Voltage) domain of Arabidopsis phototropin 2 ⁶. Upon exposure to blue light (~450 nm), miniSOG generates ROS including singlet oxygen with flavin mononucleotide as a cofactor^6-8, which is present in all cells. We constructed a His-mSOG fusion protein by tagging miniSOG to the C-terminus of histone-72, a C. elegans variant of Histone 3. We generated a single-copy transgene⁹ to express His-mSOG in the germline of C. elegans. Under normal culture conditions in the dark, the His-mSOG transgenic worms have normal brood size and life span⁴. Upon exposure to blue LED light, the His-mSOG worms produce heritable mutations among their progeny⁴. The spectrum of induced mutations includes nucleotide changes, such as G:C to T:A and G:C to C:G, and small chromosome deletions⁴. This mutagenesis procedure is simple to perform, and requires minimal lab safety setup. Here, we describe the LED illumination setup and procedures for optogenetic mutagenesis.

Protocol

1. Construction of the LED Illuminator NOTE: The required LED equipment is summarized in the List of Materials. The entire LED setup is small and can be placed anywhere in the lab, although we recommend it be placed in a dark room to control light exposure of worms4. Connect the LED to a controller with cables (Figure 1A, D). Connect the controller to a digital function generator/amplifier with a BNC cable (Figure 1A, C, D). Fix the LED light 10 cm above the stage using a custom holder (Figure 1A). NOTE: We made the holder with metal parts. Cover the LED illumination setup partially, but avoid heat accumulation (Figure 1B), which makes worms sick. NOTE: We use a custom-made hard plastic cover with open top and bottom (Figure 1A). The cover is not required for the mutagenesis itself, but is used to limit unnecessary exposure of the blue light to the surroundings. We advise to not cover the LED setup entirely (Figure 1B) to prevent overheating the worms. Wear laser-protective glasses. NOTE: We wear laser-protective glasses because the light is very bright. Sunglasses may work as well. Switch the lever of the LED controller to 'Int.' for continuous illumination (Figure 1D) and set it at 65% of the maximum power (Figure 1C). Use a photometer to measure the blue light intensity on the stage where the worms are placed using manufacturer's protocol. The setup gives 2.0 mW/mm2 under continuous illumination. 2. Blue Light Treatment NOTE: Use the strain CZ20310 juSi164[Pmex-5-HIS-72::miniSOG-3'UTR(tbb-2) + Cb-unc-119(+)] unc-119(ed3)III4. The strain is available at the Caenorhabditis Genetics Center (CGC, http://www.cgc.cbs.umn.edu/). Maintain the His-mSOG strain in the dark on standard nematode growth medium (NGM) plates with E. coli OP50 following a standard protocol10,11. NOTE: Under ambient light, juSi164-containing strains do not display a mutation rate above a spontaneous rate4. Routine strain passage using a dissecting scope with a halogen lamp generally does not cause mutations as well. Nonetheless, care should be taken to avoid unnecessary exposure to light, e.g., keeping the strains in a standard dark-inside incubator, or covering the strains with foil. It is advised to freeze aliquots of the strain after receiving it in case unnecessary mutations accumulate over time. Pick gravid young adult (approximately 12 h post-L4) animals with a worm pick11. NOTE: Younger animals show less mutagenic effect4, while older animals may have lower brood size. Cut a filter paper to fit a 60 mm plate with a 25 mm x 25 mm square hole and place onto an unseeded plate (Figure 1E). NOTE: A square punch can be used but the size of the punch does not have to be precise. Drop 100 µL of 100 mM CuCl2 on the filter paper evenly to restrict worms within the square hole on the plate. Pick desired number of gravid young adult hermaphrodites (see Step 3 for an example) and transfer to the center of the CuCl2 plate. Wear laser protective glasses. Switch ON the LED and the function generator. Switch the lever of the LED controller to 'Ext.' to control it by the function generator (Figure 1D). Set the controller at 65% of the maximum power and the function generator as sine wave, 4 Hz (Figure 1C). Place the CuCl2 plate from Step 2.5 without a lid on the stage under the LED (Figure 1A). Illuminate the animals with blue light for 30 min. Transfer healthy looking worms to a seeded plate as P0. NOTE: The desired number of P0 depends on the type of screen. See Step 3 for an example. The P0 worms may appear to be uncoordinated immediately after light treatment and will recover over time. Keep worms covered using foil in an incubator or in the dark; and follow the standard procedure to begin screening desired phenotypes among their progeny2,10. 3. Example of a Forward Genetic Screen NOTE: This is an example of the clonal screen with 120 haploid genomes to check if the LED and strain are working. Figure 2 shows a schematic of the procedure. [Day1] After blue light treatment, pick five healthy looking worms onto a NGM plate with OP50 as P0, keep them in a 20 °C incubator, and let them lay eggs for 1 d ('Day1' plate). [Day2] Transfer P0 onto a new seeded plate ('Day2' plate). [Day3] Pick and kill P0 on the 'Day2' plate by burning them. [Day4] Pick 30 gravid young adult F1 from the 'Day1' plate and transfer them onto individual plates (30 F1 plates for 'Day1'). [Day5] Repeat Step 3.4 for the 'Day2' plate (30 F1 plates for 'Day2'). [Day7-9] Check the abnormal morphologies and/or behavior of F2 worms compared to the WT strain (Figure 3A) under a standard stereomicroscope when they become adults. NOTE: A heritable mutation displays about a quarter of worms with the same phenotype among F2 when it is recessive with high penetrance. However, some mutants may grow slowly and/or show partial penetrance. Therefore, it is possible that less than a quarter of the mutants can be found on a plate. Pick worms with visible phenotypes individually and check the heritability of the phenotype in the next generation. 4. Example of an Extrachromosomal Transgene Integration Inject the DNA of interest into CZ20310 juSi164[Pmex-5-HIS-72::miniSOG-3'UTR(tbb-2) + Cb-unc-119(+)] unc-119(ed3) and prepare transgenic worms with a low transmission rate (10-30%) following a standard injection protocol12,13. NOTE: Alternatively, established extrachromosomal arrays can be crossed into the juSi164 strains. To avoid genotyping for juSi164, juSi164 heterozygotes may be used as the P0 although efficiency might be decreased. The genotyping method for juSi164 is described in Section 5. [Day1] Treat ~20 transgenic animals as P0 with blue light as described in Step 2. NOTE: More P0 worms are necessary depending on the transmission rate. Transgenes can be identified by phenotypes or coinjection marker12,13. Pick 10 healthy looking P0 onto individual plates, keep them in a 20 °C incubator, and let them lay eggs for a few days (P0 plates) [Day4] Single out 20 transgenic F1 worms from each P0 plate onto individual plates and let them lay eggs. (20 F1 x 10 P0 plates= 200 F1 plates in total.) [Day7] Find F1 plates with >75% F2 having transgenes. Pick five transgenic animals from the >75% transmission rate F1 plates (F2 plates). [Day10] Keep F2 plates with 100% transmission rate as potential integrated lines. Outcross the potential integrated lines using a standard procedure10 to check the Mendelian segregation and to remove potential background mutations. 5. Genotyping Lyse 20 outcrossed F2 worms in lysis buffer using a standard protocol14. Perform PCR using the primers for MosSCI insertion9, juSi164, (Table 1) with an annealing temperature of 58 °C following a standard protocol14. NOTE: It is unnecessary to genotype unc-119(ed3) because unc-119(ed3) can be detected by uncoordinated behavior after removing the rescuing transgene (juSi164) in the following Steps 5.4 and 5.5. Run agarose gel electrophoresis15 and examine the band sizes (Table 1) to find the WT for juSi164. Examine if the F2 progeny has paralyzed worms due to the existence of unc-119(ed3). If uncoordinated worms are found on step 5.4, single several nonuncoordinated worms to obtain all nonuncoordinated worms in the next generation.

Representative Results

We performed a forward genetic screen with CZ20310 juSi164 unc-119(ed3) using 30 min light exposure following the standard protocol described in Sections 2 and 3. Among 60 F1 plates corresponding to 120 mutagenized haploid genomes, we found 8 F1 plates with F2 worms displaying visible phenotypes such as body size defect (Figure 3B), uncoordinated movement (Figure 3C), and adult lethality (Figure 3D</strong…

Discussion

Here, we describe a detailed procedure of optogenetic mutagenesis using His-mSOG expressed in the germline and provide an example of a small scale forward genetic screen. Compared to standard chemical mutagenesis, this optogenetic mutagenesis method has several advantages. Firstly, it is nontoxic, thereby keeping lab personnel away from toxic chemical mutagens such as EMS, ENU and trimethylpsoralen with ultraviolet light (TMP/UV). Secondly, the experimental procedure of mutagenesis can be used for a small number of the P…

Materials

Ultra High Power LED light source	Prizmatix	UHP-mic-LED-460	460 ± 5 nm
LED controller	Prizmatix	UHPLCC-01
Digital function generator/amplifier	PASCO	PI-9587C	PI-9587C is no longer available. The replacement is PI-8127.
BNC cable male/male	THORLABS	CA3136
USB-TTL interface	Prizmatix		Optional
Photometer	THORLABS	PM50 and Model D10MM
Filter paper	Whatman	1001-110
Copper chloride dihydrate (CuCl2∙2H2O)	Sigma	C6641
Stereomicroscope	Leica	MZ95
NGM plate			Dissolve 5g NaCl, 2.5g Peptone, 20g Agar, 10 µg/ml cholesterol in 1 L H2O. After autoclaving, add 1 mM CaCl2, 1 mM MgSO4, 25 mM KH2PO4(pH6.0).
Lysis solution			10 mM Tris(pH8.8), 50 mM KCl, 0.1% Triton X100, 2.5 mM MgCl2, 100 µg/ml Proteinase K