Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

Summary

The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these "morphants" by co-injection of mRNA encoding this gene product.

Abstract

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

Introduction

The utilization of zebrafish as a model for the study of cardiovascular development and disease offers a variety of advantages, including high conservation of gene function, optical transparency, rapid cardiovascular development, and cheaper cost when compared to traditional in vivo models¹. Morpholino oligonucleotides (MOs) are the most commonly used antisense gene knockdown tools for the zebrafish model. MOs are frequently used to determine a phenotype or to probe gene function. Dr. James Summerton initially developed the morpholino delivery system for the in vivo inhibition of mRNA translation as an attempt to develop thera....

Protocol

All experiments were carried out in accordance with the accepted standards of humane animal care under the regulation of the IACUC at QU; animals were held in the zebrafish facility under Qatar University Biomedical Research Center (QU-BRC). All animals used in these experimental studies were under 3 days post-fertilization (dpf).

NOTE: For each experimental group, it is advisable to use at least 30 embryos for statistical rigor. The experimental groups are as follows:

Control group: This group includes embryos cultured in egg water without any injections. Results here will form the control basel....

Results

The graph in Figure 6 illustrates the average percent of embryos surviving at 24, 48, and 72 hpf for both HAND2- specific MO and control scrambled MO-injected embryos. The 1 mM (8 ng/µL) and 0.8 mM (6.4 ng/µL) MO-injected embryos showed a significant reduction in survival percentage compared to control scrambled MO-injected embryos. This was observed across each measured time point where lethality or malformation was observed. The results indicated that a high concentration of HAND2MO had a .......

Discussion

Morpholino (MO) technology has been extensively used in zebrafish, xenopus, sea urchins, and more recently in cell culture model systems. With most methods, along with the benefits, there are also pitfalls that the experimenter should be aware of. One of the major pitfalls with MO technology includes the concern that phenotypic effects observed by the MO-mediated gene knockdown approach are not due to the loss of function associated with the primary gene product but that some other genes along with the primary gene or in.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acrylamide 40%	Sigma	Sigma, cat. no. C977M88
Agarose	Sigma-Aldrich	Sigma-Aldrich cat. no A9539-250G
All Prep DNA/RNA Mini Kit	Qiagen	Qiagen cat. no. 80204.
alpha Tubulin	Abcam	Abcam- ab4074	Rabbit polyclonal to alpha Tubulin lot GR3 180877-1 (50 kDa)
Ammonium persulfate molecular grade	Sigma	Sigma, cat. no C991U65
BV10 capillary beveller	Sutter Instruments Product	Sutter Instruments Product Catalog # BV10
Chemiluminescence Imaging Gene Gnome	SYNGENE	SYNGENE
Cleaver Scientific Blotting	CVS10D_OmniPAGEMini	CVS10D_OmniPAGEMini
Coomassie	Thermo Fisher	Thermo Fisher cat. no C861C44
Electrochemiluminescence (ECL) kit	Abcam Biochemicals	Abcam Biochemicals cat. no ab65623
Glycine	Sigma	Sigma, cat. no C988U91
Goat anti Rabbit	Abcam	Abcam-  ab6721	Goat Anti-Rabbit IgG H&L (HRP) 2nd antibodies lot GR3179871-1
HAND2	Gene tools	Custom made for HAND2 (NM_021973)	5'-CCTCCAACTAAACTCATGGCGAC AG-3'
Hand2	Abcam	Abcam- ab10131	Rabbit polyclonal Anti-HAND2 antibody lot GR143200-9 (24- 26 kDa)
HAND2 (NM_021973) Human Tagged ORF Clone	OriGene Technologies, Inc	RC224436L3	Vector: pLenti-C-Myc-DDK-P2A-Puro (PS100092)
IBI DNA/RNA/Protein Extraction Kit	IBI Scientific	IBI Scientific cat. no -r IB47702
Imaging System	iBright	iBright CL1000 Imaging System
Isopropanol	Sigma-Aldrich	Sigma-Aldrich cat. no 278475-2L
Laemmli sample loading buffer (4x)	Sigma-Aldrich	Sigma-Aldrich cat. no 70607
Mercaptoethanol	Sigma	Sigma, cat. no M6250-1L
Microplate Spectrophotometer with the Gen5 Data Analysis software interface	Epoch	Epoch
Microscope	Ziess SteREO Lumar V12 Flourescence Microscope	Ziess SteREO Lumar V12 Flourescence Microscope
Mineral oil	Fisher Scientific	Fisher Scientific cat. no 0121-1
mMESSAGE mMACHINE T7/T3/SP6 Transcription Kit	Thermo Fisher	Thermo Fisher cat. no.AM1340	for mRNA generation
Nuclease-free water	New England Biolabs	New England Biolabs cat. no B1500L
PC-100 Micropipette Puller	NARISHIGE GROUP Product	NARISHIGE GROUP Product Catalog # PC-100
Phenol red	Sigma	Sigma, cat. no. P-0290
Picolitre Injector	Harvard Apparatus	Harvard Apparatus cataloge # PLI-90A
Pierce Bicinchoninic acid assay (BCA) Protein Assay kit	Thermo Fisher	Thermo Fisher cat. no 23227
PMSF, Protease inhibitor as protease inhibitors	Thermo Fisher	Thermo Fisher cat. no 36978
Ponceau S	Sigma-Aldrich	Sigma-Aldrich cat. no 10165921001
Protease Inhibitor Cocktail	Thermo Fisher	Thermo Fisher cat. no 88668
Protein ladder	SMOBiO	SMOBiO cat. no PM2500
Radioimmunoprecipitation Assay (RIPA)	Thermo Fisher	Thermo Fisher cat. no 89900
Ringer’s solution	Thermofisher	Catalog No.S25513
SDS	Sigma	Sigma, cat. no 436143
Standard Control	Gene tools	SKU: PCO-StandardControl-100	5'-CCTCTTACCTCAGTTACAATTTAT A-3'- that targets a human beta-globin intron mutation
Stripping buffer	Sigma-Aldrich	Sigma-Aldrich cat. 21059
Temed	IBI scientific	IBI scientific cat. no C000A52
Tris Base	Thermo Fisher	Thermo Fisher cat. no BP-152-500
Tween	sigma life science	sigma life science cat. no P2287
Zebra Box Revolution-Danio Track system chamber with the EthoVision XT 11.5 software	Noldus Information Technology, NL	Noldus Information Technology, NL
Zeiss Axiocam ERc 5s	Zeiss	Stemi 508 Zeiss
Zeiss Stemi 2000-C	Zeiss	Stemi 2000-C