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