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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 (Gja1M213L/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 vitro1,2,3.

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 algorithm4<.......

Representative Results

The CRISPR/Cas9 gene-editing system produces an ATG to TTA mutation and a silent TCC to TCG mutation at 56,264,279 to 56,264,281 and at 56,264,291 to 56,264,293 on mouse chromosome 10, or at 869 to 871 and 881 to 883 on Gja1 mRNA, respectively. Those mutation results in a Methionine 213 to Leucine (M213L) mutation on GJA1 protein and the TTC to TTG mutation disrupts a nearby PAM sequence to avoid undesired gene edition (Figure 1). The details of the mutation are described in a previous repor.......

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.......

Acknowledgements

The project was supported by National Institutes of Health grants (R01HL152691, R01HL138577, and R01HL159983) to RMS.

....

Materials

NameCompanyCatalog NumberComments
0.2 mL 8-Strip PCR tubeThomas Scientific1148A28
0.5 M EDTA pH 8.0Invitrogen15575-038For pronuclear micorinjection buffer
10x CutSmart bufferNew England BiolabsB7204SDigestion buffer for NlaIII
1 M Tris-HCl pH 7.5Invitrogen15567-027For pronuclear micorinjection buffer
50x TAE BufferInvitrogen24710-030
96-well Thermal cyclerApplied BiosystemsModel # 9902
AgaroseSigma-AldrichA9539
C57BL/6JThe Jackson Laboratory664mouse strain
Chemidoc MP imaging systemBio-radTo take gel image by 302 nm UV light
eSpCas9 proteinMilliporeSigmaESPCAS9PRO-50UG
Gel Lading Dye Purple (6x)New England BiolabsB7024SLoading buffer for electrophoresis
Gloves
hCG (human chorionic gonadotropin)MilliporeSigma23-073-425G
HyaluronidaseMilliporeSigmaH3884-100MG
Image Lab softwareBio-radTo analyze gel image
Inverted stereo microscope whit plasDICZeissAxiovert A1
KAPA Mouse Genotyping KitsRoche DiagnosticsKK7352For tissue or cell lysis, DNA extraction, and PCR master mix
KSOMLifeGlobalZEKS-050embryo culture medium
Lab coart
M2 mediumMilliporeSigmaMR015D
NlaIIINew England BiolabsR0125STo digest PCR product
Nuclease-Free WaterAmbionAM9937To dilute reagents
Paraffin oilNacalai USA2613785
Plugged 20 μL fine pipette tipFisherScientific02-707-171To pick up blastocytes
PMSG (pregnant mare’s serum gonadotropin)ProSpec-TanyHOR-272
Sodium ChlorideInvitrogenAM9760GFor pronuclear micorinjection buffer
SYBR safe DNA Gel StainInvitrogenS33102To detect bands in gel
tracrRNADharmaconU-002000-120Guid RNA

References

  1. Smyth, J. W., Shaw, R. M. Autoregulation of connexin43 gap junction formation by internally translated isoforms. Cell Reports. 5 (3), 611-618 (2013).
  2. Basheer, W. A., et al. GJA1-20k arranges....

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