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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Developmental Biology

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

Published: May 13th, 2016

DOI:

10.3791/54055

1Department of Molecular and Cell Biology, University of California, Berkeley

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

One fundamental component of understanding how biodiversity arises is determining the genetic and developmental bases of evolved phenotypic changes in nature. The threespine stickleback fish, Gasterosteus aculeatus, has emerged as an excellent model for studying the genetic basis of evolution. Sticklebacks have undergone many adaptive evolutionary changes as marine fish have colonized countless freshwater environments around the northern hemisphere, resulting in dramatic morphological, physiological, and behavioral changes1. The genomes of individuals from twenty-one stickleback populations have been sequenced and assembled, and a high density link....

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All fish work was approved by the Institutional Animal Care and Use Committee of the University of California-Berkeley (protocol number R330).

1. Prepare Nucleic Acids for Injection

  1. Tol2 Plasmid Transgenesis (Adapted from Fisher26).
    1. Cut 10 µg transposase plasmid (pCS-Tp)39 with 10 U NotI in supplied buffer for 1 hr at 37 °C to linearize.
      Note: Material Transfer Agreements may be required to obtain Tol2 plasmids.
    2. Extract the cut plasmid with.......

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For reporter gene transgenes that have enhancer activity, successful injection will result in specific, cellular expression of the transgene (Figure 4A, 4C). Injected fish can then be outcrossed to produce stable lines (example of a BAC stable line shown in Figure 4B). Injecting DNA into stickleback embryos typically results in far higher lethality than RNA alone. It is typical to see up to 50% (sometimes even more) lethality or malformat.......

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Injecting one-cell stickleback embryos for transgenesis or genome editing presents three main challenges. First, relative to zebrafish embryos, the stickleback embryonic chorion is tough and will often break needles. This problem can be partially overcome by using thicker and stronger glass micropipettes and injecting perpendicular to the chorion (see Protocol, Figure 2). Ensuring that as little water as possible is added to the embryos (just enough to cause the chorion to swell and lift away from the ce.......

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This work was funded in part by NIH R01 #DE021475 (CTM), an NIH Predoctoral Training Grant 5T32GM007127 (PAE), and an NSF Graduate Research Fellowship (NAE). We thank Kevin Schwalbach for performing BAC recombineering and injections, Nick Donde for generating CRISPR Sanger sequencing data, and Katherine Lipari for helpful feedback on the injection protocol.

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Name Company Catalog Number Comments
Stereomicroscope with transillumination Leica S6e/ KL300 LED
Manual micromanipulator Applied Scientific Instrumentation MM33 Marzhauser M33 Micromanipulator
Pressure Injecion system Applied Scientific Instrumentation MPPI-3
Back pressure unit Applied Scientific Instrumentation BPU
Micropipette holder kit Applied Scientific Instrumentation MPIP
Magnetic base holder Applied Scientific Instrumentation Magnetic base
Foot switch Applied Scientific Instrumentation FSW
Iron plate (magnetic base) Narishige IP
Flaming/Brown Micropipette Puller Sutter Instrument P-97
Disposable transfer pipettes Fisher 13-711-7M
0.5% phenol red in DPBS Sigma  P0290 injection tracer
#5 forceps, biologie dumoxel  Fine Science Tools 11252-30 for needle breaking
Micropipette Storage Jar World Precision Instruments E210 holds needles
6", 6 teeth per inch plaster drywall saw Lenox 20571 (S636RP) hold eggs for injection
13 cm x 13 cm glass plate any hardware store -
Borosilicate glass capillaries, 1.0 mm OD/0.58 mm ID  World Precision Instruments 1B100-F4 *harder glass than zebrafish injection capillaries
150 x 15mm petri dish Fisher FB0875714 raise stickleback embryos
35 x 10mm petri dish Fisher 08-757-100A store eggs pre-injection
Instant Ocean Salt Instant Ocean SS15-10
Sodium Bicarbonate Sigma S5761-500G
Tricaine methanesulfonate/MS-222 Western Chemical Inc MS222 fish anaesthesia/euthanasia
Sp6 transcription kit Ambion AM1340 For transcription of TALENs and transposase mRNA
RNeasy cleanup kit Qiagen 74104 purify transposase or TALEN RNA
QiaQuick PCR cleanup kit Qiagen 28104 clean up plasmids for injection
Proteinase K 20 mg/ml Ambion AM2546 for DNA preparation
Nucleobond BAC 100 kit Clontech 740579 for BAC DNA preparation
NotI NEB R0189L
Phusion polymerase Fisher F-530L
Qiagen PlasmidPlus Midi kit Qiagen 12943 contains endotoxin rinse buffer
QIAQuick Gel Extraction Qiagen 28704  for sequencing induced mutations
Phenol:chloroform:Isoamyl alcohol Sigma P2069-100ML
Sodium acetate Sigma S2889-250G
Ethanol (molecular biology grade) Sigma E7023-500ML
Agarose Sigma A9539
50X Tris-acetate-EDTA buffer ThermoFisher B49
0.5-10KbRNA ladder ThermoFisher 15623-200
Nanodrop  Spectrophotometer Thermo Scientific Nanodrop 2000
Paraformaldehyde Sigma 158127-500G
10X PBS ThermoFisher 70011-044
1kb Plus DNA Ladder ThermoFisher 10787-018
Potassium Chloride Sigma P9541-500G
Magnesium Chloride Sigma M8266-100G
NP-40 ThermoFisher 28324
Tween 20 Sigma P1379-500ML
Tris pH 8.3 Teknova T1083
12-strip PCR tube Thermo Scientific AB-1113

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