CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) ...mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20–33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as
EGFP
,
mCherry
,
Cre
, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.
Immunodeficient animals are valuable models for the engraftment of exogenous tissues; they are widely used in many fields, including the creation of humanized animal models, as well as regenerative ...medicine and oncology. Compared with mice, laboratory rats have a larger body size and can more easily undergo transplantation of various tissues and organs. Considering the absence of high-quality resources of immunodeficient rats, we used the CRISPR/Cas9 genome editing system to knock out the interleukin-2 receptor gamma chain gene (
Il2rg
) in F344/Jcl rats—alone or together with recombination activating gene 2 (
Rag2
)—to create a high-quality bioresource that researchers can freely use: severe combined immunodeficiency (SCID) rats. We selected one founder rat with frame-shift mutations in both
Il2rg
(5-bp del) and
Rag2
(1-bp del+2-bp ins/7-bp del+2-bp ins), then conducted mating to establish a line of immunodeficient rats. The immunodeficiency phenotype was preliminarily confirmed by the presence of severe thymic hypoplasia in
Il2rg
-single knockout (sKO) and
Il2rg
/
Rag2
-double knockout (dKO) rats. Assessment of blood cell counts in peripheral blood showed that the white blood cell count was significantly decreased in sKO and dKO rats, while the red blood cell count was unaffected. The decrease in white blood cell count was mainly caused by a decrease in lymphocytes. Furthermore, analyses of lymphocyte populations via flow cytometry showed that the numbers of B cells (CD3
-
CD45
+
) and natural killer cells (CD3
-
CD161
+
) were markedly reduced in both knockout rats. In contrast, T cells were markedly reduced but showed slightly different results between sKO and dKO rats. Notably, our immunodeficient rats do not exhibit growth retardation or gametogenesis defects. This high-quality SCID rat resource is now managed by the National BioResource Project in Japan. Our SCID rat model has been used in various research fields, demonstrating its importance as a bioresource.
CRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive.
Here, we show that the electroporation of Cas9, two gRNAs, ...and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats.
The high efficiency of CLICK provides homozygous knock-ins in oocytes carrying tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice.
Cell groups containing catecholamines provide a useful model to study the molecular and cellular mechanisms underlying the morphogenesis, physiology, and pathology of the central nervous system. For ...this purpose, it is necessary to establish a system to induce catecholaminergic group-specific expression of Cre recombinase. Recently, we introduced a gene cassette encoding 2A peptide fused to Cre recombinase into the site between the C-terminus and translational termination codons of the rat tyrosine hydroxylase (TH) open reading frame by the Combi-CRISPR technology, which is a genomic editing method to enable an efficient knock-in (KI) of long DNA sequence into a target site. However, the expression patterns of the transgene and its function as well as the effect of the mutation on the biochemical and behavioral phenotypes in the KI strains have not been characterized yet.
We aimed to evaluate the usefulness of TH-Cre KI rats as an experimental model for investigating the structure and function of catecholaminergic neurons in the brain.
We detected cell type-specific expression of Cre recombinase and site-specific recombination activity in the representative catecholaminergic groups in the TH-Cre KI rat strains. In addition, we measured TH protein levels and catecholamine accumulation in the brain regions, as well as motor, reward-related, and anxiety-like behaviors, indicating that catecholamine metabolism and general behavior are apparently normal in these KI rats.
TH-Cre KI rat strains produced by the Combi-CRISPR system offer a beneficial model to study the molecular and cellular mechanics for the morphogenesis, physiology, and pathology of catecholamine-containing neurons in the brain.
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•Knock-in transgenic rats have a gene encoding a TH-2A-Cre protein precursor.•These rats showed efficient expression of recombinase in a cell type-specific manner.•The knock-in rats exhibited the normal biochemical and behavioral phenotypes.•The rats offer a model to study the structure and function of catecholamine neurons.
Abstract
Hydrogen sulfide (H
2
S) and polysulfides (H
2
S
n
, n ≥ 2) are signaling molecules produced by 3-mercaptopyruvate sulfurtransferase (3MST) that play various physiological roles, including ...the induction of hippocampal long-term potentiation (LTP), a synaptic model of memory formation, by enhancing N-methyl-
d
-aspartate (NMDA) receptor activity. However, the presynaptic action of H
2
S/H
2
S
n
on neurotransmitter release, regulation of LTP induction, and animal behavior are poorly understood. Here, we showed that H
2
S/H
2
S
2
applied to the rat hippocampus by in vivo microdialysis induces the release of GABA, glutamate, and
d
-serine, a co-agonist of NMDA receptors. Animals with genetically knocked-out 3MST and the target of H
2
S
2
, transient receptor potential ankyrin 1 (TRPA1) channels, revealed that H
2
S/H
2
S
2
, 3MST, and TRPA1 activation play a critical role in LTP induction, and the lack of 3MST causes behavioral hypersensitivity to NMDA receptor antagonism, as in schizophrenia. H
2
S/H
2
S
n
, 3MST, and TRPA1 channels have therapeutic potential for psychiatric diseases and cognitive deficits.
GABA is a major neurotransmitter in the mammalian central nervous system. Glutamate decarboxylase (GAD) synthesizes GABA from glutamate, and two isoforms of GAD, GAD65, and GAD67, are separately ...encoded by the Gad2 and Gad1 genes, respectively. The phenotypes differ in severity between GAD single isoform‐deficient mice and rats. For example, GAD67 deficiency causes cleft palate and/or omphalocele in mice but not in rats. In this study, to further investigate the functional roles of GAD65 and/or GAD67 and to determine the contribution of these isoforms to GABA synthesis during development, we generated various kinds of GAD isoform(s)‐deficient rats and characterized their phenotypes. The age of death was different among Gad mutant rat genotypes. In particular, all Gad1−/−; Gad2−/− rats died at postnatal day 0 and showed little alveolar space in their lungs, suggesting that the cause of their death was respiratory failure. All Gad1−/−; Gad2−/− rats and 18% of Gad1−/−; Gad2+/− rats showed cleft palate. In contrast, none of the Gad mutant rats including Gad1−/−; Gad2−/− rats, showed omphalocele. These results suggest that both rat GAD65 and GAD67 are involved in palate formation, while neither isoform is critical for abdominal wall formation. The GABA content in Gad1−/−; Gad2−/− rat forebrains and retinas at embryonic day 20 was extremely low, indicating that almost all GABA was synthesized from glutamate by GADs in the perinatal period. The present study shows that Gad mutant rats are a good model for further defining the role of GABA during development.
Arrhythmogenic cardiomyopathy (ACM) caused by TMEM43 p.S358L is a fully penetrant heart disease that results in impaired cardiac function or fatal arrhythmia. However, the molecular mechanism of ACM ...caused by the TMEM43 variant has not yet been fully elucidated. In this study, we generated knock‐in (KI) rats harboring a Tmem43 p.S358L mutation and established induced pluripotent stem cells (iPSCs) from patients based on the identification of TMEM43 p.S358L variant from a family with ACM. The Tmem43‐S358L KI rats exhibited ventricular arrhythmia and fibrotic myocardial replacement in the subepicardium, which recapitulated the human ACM phenotype. The four‐transmembrane protein TMEM43 with the p.S358L variant (TMEM43S358L) was found to be modified by N‐linked glycosylation in both KI rat cardiomyocytes and patient‐specific iPSC‐derived cardiomyocytes. TMEM43S358L glycosylation increased under the conditions of enhanced endoplasmic reticulum (ER) stress caused by pharmacological stimulation or age‐dependent decline of the ER function. Intriguingly, the specific glycosylation of TMEM43S358L resulted from the altered membrane topology of TMEM43. Moreover, unlike TMEM43WT, which is mainly localized to the ER, TMEM43S358L accumulated at the nuclear envelope of cardiomyocytes with the increase in glycosylation. Finally, our comprehensive transcriptomic analysis demonstrated that the regional differences in gene expression patterns between the inner and outer layers observed in the wild type myocardium were partially diminished in the KI myocardium prior to exhibiting histological changes indicative of ACM. Altogether, these findings suggest that the aberrant accumulation of TMEM43S358L underlies the pathogenesis of ACM caused by TMEM43 p.S358L variant by affecting the transmural gene expression within the myocardium.
Inhibitory neurotransmitter GABA is synthesized by two enzymes, the glutamate decarboxylase 67‐kDa isoform (GAD67) and the 65‐kDa isoform (GAD65). Knocking out the Gad1 gene, which encodes GAD67, is ...lethal in mice, and therefore, the association between Gad1 and emotional function was unclear. In the present study, we assessed the impact of Gad1 knockout on fear expression and fear memory for the first time, using newly generated knockout rats.
The GABAergic system is thought to play an important role in the control of cognition and emotion, such as fear, and is related to the pathophysiology of psychiatric disorders. For example, the expression of the 67‐kDa isoform of glutamate decarboxylase (GAD67), a GABA‐producing enzyme, is downregulated in the postmortem brains of patients with major depressive disorder and schizophrenia. However, knocking out the Gad1 gene, which encodes GAD67, is lethal in mice, and thus, the association between Gad1 and cognitive/emotional functions is unclear. We recently developed Gad1 knockout rats and found that some of them can grow into adulthood. Here, we performed fear‐conditioning tests in adult Gad1 knockout rats to assess the impact of the loss of Gad1 on fear‐related behaviors and the formation of fear memory. In a protocol assessing both cued and contextual memory, Gad1 knockout rats showed a partial antiphase pattern of freezing during training and significantly excessive freezing during the contextual test compared with wild‐type rats. However, Gad1 knockout rats did not show any synchronous increase in freezing with auditory tones in the cued test. On the other hand, in a contextual memory specialized protocol, Gad1 knockout rats exhibited comparable freezing behavior to wild‐type rats, while their fear extinction was markedly impaired. These results suggest that GABA synthesis by GAD67 has differential roles in cued and contextual fear memory.
Glutamate decarboxylase 67-kDa isoform (GAD67), which is encoded by the
gene, is one of the key enzymes that produce GABA. The reduced expression of GAD67 has been linked to the pathophysiology of ...schizophrenia. Additionally, the excitatory glutamatergic system plays an important role in the development of this disorder. Animal model studies have revealed that chronic blockade of NMDA-type glutamate receptors can cause GABAergic dysfunction and long-lasting behavioral abnormalities. Based on these findings, we speculated that
haplodeficiency combined with chronic NMDA receptor blockade would lead to larger behavioral consequences relevant to schizophrenia in a rat model. In this study, we administered an NMDAR antagonist, MK-801 (0.2 mg/kg), to CRISPR/Cas9-generated
rats during adolescence to test this hypothesis. The MK-801 treated
rats showed a shorter duration in each rearing episode in the open field test than the saline-treated
rats. In contrast, immobility in the forced swim test was increased and fear extinction was impaired in
rats irrespective of MK-801 treatment. Interestingly, the time spent in the center region of the elevated plus-maze was significantly affected only in the saline-treated
rats. Additionally, the MK-801-induced impairment of the social novelty preference was not observed in
rats. These results suggest that the synergistic and additive effects of
haplodeficiency and NMDA receptor blockade during adolescence on the pathogenesis of schizophrenia may be more limited than expected. Findings from this study also imply that these two factors mainly affect negative or affective symptoms, rather than positive symptoms.
Patients with pulmonary arterial hypertension (PAH) carrying bone morphogenetic protein receptor type 2 (Bmpr2) mutations present earlier with severe hemodynamic compromise and have poorer survival ...outcomes than those without mutation. The mechanism underlying the worsening clinical phenotype of PAH with Bmpr2 mutations has been largely unaddressed in rat models of pulmonary hypertension (PH) because of the difficulty in reproducing progressive PH in mice and genetic modification in rats. We tested whether a clinically-relevant Bmpr2 mutation affects the progressive features of monocrotaline (MCT) induced-PH in rats.
A monoallelic single nucleotide insertion in exon 1 of Bmpr2 (+/44insG) was generated in rats using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9, then PH, pulmonary vascular disease (PVD) and survival after MCT injection with or without a phosphodiesterase type 5 inhibitor, tadalafil, administration were assessed.
The +/44insG rats had reduced BMPR2 signalling in the lungs compared with wild-type. PH and PVD assessed at 3-weeks after MCT injection were similar in wild-type and +/44insG rats. However, survival at 4-weeks after MCT injection was significantly reduced in +/44insG rats. Among the rats surviving at 4-weeks after MCT administration, +/44insG rats had increased weight ratio of right ventricle to left ventricle plus septum (RV/LV + S) and % medial wall thickness (MWT) in pulmonary arteries (PAs). Immunohistochemical analysis showed increased vessels with Ki67-positive cells in the lungs, decreased mature and increased immature smooth muscle cell phenotype markers in the PAs in +/44insG rats compared with wild-type at 3-weeks after MCT injection. Contraction of PA in response to prostaglandin-F2α and endothelin-1 were significantly reduced in the +/44insG rats. The +/44insG rats that had received tadalafil had a worse survival with a significant increase in RV/(LV + S), %MWT in distal PAs and RV myocardial fibrosis compared with wild-type.
The present study demonstrates that the Bmpr2 mutation promotes dedifferentiation of PA smooth muscle cells, late PVD and RV myocardial fibrosis and adversely impacts both the natural and post-treatment courses of MCT-PH in rats with significant effects only in the late stages and warrants preclinical studies using this new genetic model to optimize treatment outcomes of heritable PAH.
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