Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 a 25:24:1 mixture of phenol:chloroform:isoamyl alcohol and ethanol precipitate with sodium acetate according to standard protocols⁴⁰. Resuspend plasmi....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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 mm x 10 mm 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
0.5-10 Kb RNA ladder	ThermoFisher	15623-200
Nanodrop  Spectrophotometer	Thermo Scientific	Nanodrop 2000
Paraformaldehyde	Sigma	158127-500G
10x PBS	ThermoFisher	70011-044
1 kb 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