Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein

Summary

The present protocol describes a single M213L mutation in Gja1 that retains full-length Connexin43 generation but prevents translation of the smaller GJA1-20k internally translated isoform.

Abstract

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily relative to traditional homologous recombination. The CRISPR-Cas9 system is particularly attractive when a single-point mutation is desired. The gap junction protein, Connexin 43 (Cx43), is encoded by gene Gja1, which has a single coding exon and cannot be spliced. However, Gja1 produces not only full-length Cx43 protein but up to six N-terminus truncated isoforms by a process known as internal translation, the result of ribosomal translation initiation at internal AUG (Methionine) start sites. GJA1-20k is the most commonly generated truncated isoform of Cx43 initiated at the AUG codon at position 213 of Gja1 mRNA. Because residue 213 occurs at the end of the last transmembrane domain of Cx43, GJA1-20k is effectively the 20 kDa C-terminus tail of Cx43 as an independent protein. Previous investigators identified, in cells, that a critical role of GJA1-20k is to facilitate trafficking of full-length Cx43 gap junction hemichannels to the plasma membrane. To examine this phenomenon in vivo, a mutant mouse with a Gja1 point-mutation was generated that replaces the ATG (Methionine) at residue 213 with TTA (Leucine, M213L mutation). The result of M213L is that Gja1 mRNA and full-length Cx43 are still generated, yet the translation of Gja1-20k is significantly reduced. This report focuses on choosing the restriction enzyme site to develop a one amino acid mutated (Gja1^M213L/M213L) mouse model. This protocol describes genetically modified mice by the CRISPR-Cas9 system and rapid genotyping by combining PCR and restriction enzyme treatments.

Introduction

The full-length Connexin 43 (Cx43) and the N-terminus truncated isoform, GJA1-20k, encoded by the same GJA1 mRNA but utilize different start codons¹ to initiate translation. Cx43 translation occurs at the first AUG start codon, whereas GJA1-20k translation initiates at the AUG at residue 213. It was previously found that GJA1-20k has essential roles for full-length Cx43 trafficking, actin stabilization, and regulation of mitochondrial morphology in vitro^1,2,3.

To understand the role of GJA1-20k in vivo, a G....

Protocol

`All animal care and study protocols were approved by the Institutional Animal Care and Use Committees of Cedars-Sinai Medical Center and the University of Utah. C57BL/6J female mice obtained from commercial sources at 8-9 weeks of age (see Table of Materials) were used for the experiments.

1. Preparation for gene targeting

1. Select the guide target sequence around the targeted mutation site coding M213 using CRISPOR web algorithm^4<.......

Discussion

A gene-modified mouse model is a common approach for understanding gene function. However, since the GJA1-20k internally translated isoform is translated from the same Gja1 mRNA as full-length Cx43, a creative strategy was devised to retain full-length Cx43 expression yet suppress GJA1-20k expression. The approach is based on a mutation of the internal start codon of GJA1-20k. With a single point mutation, M213 was switched to L on Gja1 mRNA, which succeeded in suppressing GJA1-20k expression but retain.......

Materials

Name	Company	Catalog Number	Comments
0.2 mL 8-Strip PCR tube	Thomas Scientific	1148A28
0.5 M EDTA pH 8.0	Invitrogen	15575-038	For pronuclear micorinjection buffer
10x CutSmart buffer	New England Biolabs	B7204S	Digestion buffer for NlaIII
1 M Tris-HCl pH 7.5	Invitrogen	15567-027	For pronuclear micorinjection buffer
50x TAE Buffer	Invitrogen	24710-030
96-well Thermal cycler	Applied Biosystems	Model # 9902
Agarose	Sigma-Aldrich	A9539
C57BL/6J	The Jackson Laboratory	664	mouse strain
Chemidoc MP imaging system	Bio-rad		To take gel image by 302 nm UV light
eSpCas9 protein	MilliporeSigma	ESPCAS9PRO-50UG
Gel Lading Dye Purple (6x)	New England Biolabs	B7024S	Loading buffer for electrophoresis
Gloves
hCG (human chorionic gonadotropin)	MilliporeSigma	23-073-425G
Hyaluronidase	MilliporeSigma	H3884-100MG
Image Lab software	Bio-rad		To analyze gel image
Inverted stereo microscope whit plasDIC	Zeiss	Axiovert A1
KAPA Mouse Genotyping Kits	Roche Diagnostics	KK7352	For tissue or cell lysis, DNA extraction, and PCR master mix
KSOM	LifeGlobal	ZEKS-050	embryo culture medium
Lab coart
M2 medium	MilliporeSigma	MR015D
NlaIII	New England Biolabs	R0125S	To digest PCR product
Nuclease-Free Water	Ambion	AM9937	To dilute reagents
Paraffin oil	Nacalai USA	2613785
Plugged 20 μL fine pipette tip	FisherScientific	02-707-171	To pick up blastocytes
PMSG (pregnant mare’s serum gonadotropin)	ProSpec-Tany	HOR-272
Sodium Chloride	Invitrogen	AM9760G	For pronuclear micorinjection buffer
SYBR safe DNA Gel Stain	Invitrogen	S33102	To detect bands in gel
tracrRNA	Dharmacon	U-002000-120	Guid RNA