A zebrafish tufm mutant model for the COXPD4 syndrome of aberrant mitochondrial function.

Mitochondrial dysfunction is a critical factor leading to a wide range of clinically heterogeneous and often severe disorders due to its central role in generating cellular energy. Mutations in the TUFM gene are known to cause combined oxidative phosphorylation deficiency 4 (COXPD4), a rare mitochondrial disorder characterized by a comprehensive quantitative deficiency in mitochondrial respiratory chain (MRC) complexes. The development of a reliable animal model for COXPD4 is crucial for elucidating the roles and mechanisms of TUFM in disease pathogenesis and benefiting its medical management. In this study, we construct a zebrafish tufm-/- mutant that closely resembles the COXPD4 syndrome, exhibiting compromised mitochondrial protein translation, dysfunctional mitochondria with oxidative phosphorylation (OXPHOS) defects, and significant metabolic suppression of the tricarboxylic acid (TCA) cycle. Leveraging this COXPD4 zebrafish model, we comprehensively validate the clinical relevance of TUFM mutations and identify probucol as a promising therapeutic approach for managing COXPD4. Our data offer valuable insights for understanding mitochondrial diseases and developing effective treatments.

Systematic genome editing of the genes on zebrafish Chromosome 1 by CRISPR/Cas9.

Genome editing by the well-established CRISPR/Cas9 technology has greatly facilitated our understanding of many biological processes. However, a complete whole-genome knockout for any species or model organism has rarely been achieved. Here, we performed a systematic knockout of all the genes (1333) on Chromosome 1 in zebrafish, successfully mutated 1029 genes, and generated 1039 germline-transmissible alleles corresponding to 636 genes. Meanwhile, by high-throughput bioinformatics analysis, we found that sequence features play pivotal roles in effective gRNA targeting at specific genes of interest, while the success rate of gene targeting positively correlates with GC content of the target sites. Moreover, we found that nearly one-fourth of all mutants are related to human diseases, and several representative CRISPR/Cas9-generated mutants are described here. Furthermore, we tried to identify the underlying mechanisms leading to distinct phenotypes between genetic mutants and antisense morpholino-mediated knockdown embryos. Altogether, this work has generated the first chromosome-wide collection of zebrafish genetic mutants by the CRISPR/Cas9 technology, which will serve as a valuable resource for the community, and our bioinformatics analysis also provides some useful guidance to design gene-specific gRNAs for successful gene editing.

Zebrafish microRNA miR-210-5p inhibits primitive myelopoiesis by silencing foxj1b and slc3a2a mRNAs downstream of gata4/5/6 transcription factor genes.

Zebrafish gata4/5/6 genes encode transcription factors that lie on the apex of the regulatory hierarchy in primitive myelopoiesis. However, little is known about the roles of microRNAs in gata4/5/6-regulated processes. Performing RNA-Seq deep sequencing analysis of the expression changes of microRNAs in gata4/5/6-knockdown embryos, we identified miR-210-5p as a regulator of zebrafish primitive myelopoiesis. Knocking down gata4/5/6 (generating gata5/6 morphants) significantly increased miR-210-5p expression, whereas gata4/5/6 overexpression greatly reduced its expression. Consistent with inhibited primitive myelopoiesis in the gata5/6 morphants, miR-210-5p overexpression repressed primitive myelopoiesis, indicated by reduced numbers of granulocytes and macrophages. Moreover, knocking out miR-210 partially rescued the defective primitive myelopoiesis in zebrafish gata4/5/6-knockdown embryos. Furthermore, we show that the restrictive role of miR-210-5p in zebrafish primitive myelopoiesis is due to impaired differentiation of hemangioblast into myeloid progenitor cells. By comparing the set of genes with reduced expression levels in the gata5/6 morphants to the predicted target genes of miR-210-5p, we found that foxj1b and slc3a2a, encoding a forkhead box transcription factor and a solute carrier family 3 protein, respectively, are two direct downstream targets of miR-210-5p that mediate its inhibitory roles in zebrafish primitive myelopoiesis. In summary, our results reveal that miR-210-5p has an important role in the genetic network controlling zebrafish primitive myelopoiesis.

Verapamil suppresses cardiac alternans and ventricular arrhythmias in acute myocardial ischemia via ryanodine receptor inhibition.

T-wave alternans (TWA) is a potent arrhythmia substrate under the conditions of acute myocardial ischemia. Abnormal intracellular calcium cycling contributes to the genesis of cardiac alternans. Ryanodine receptor (RyR) is a pivotal Ca2+ cycling protein central to Ca2+ signaling in the heart. Here, we investigated the potential role of RyR in cardiac alternans and ventricular arrhythmias in acute myocardial ischemia. Transmembrane action potentials were simultaneously recorded from epicardium and endocardium together with a transmural ECG and isometric contraction force in the arterially perfused left ventricular wedge preparations. Calcium alternans were induced by incremental frequency of field stimulation in rat ventricular myocytes. TWA, mechanical alternans and ventricular arrhythmias were reproducibly induced by rapid pacing in the acute ischemic wedge preparations. Compared with control group, calcium alternans ratio and spontaneous calcium release were increased in acute ischemic myocytes. Verapamil, a phenylalkylamine calcium channel blocker, can successfully abolish spontaneous calcium release, TWA, and ventricular arrhythmias. The inhibition effect of verapamil could be diminished by low concentration of ryanodine (10 nmol/L). However, nifedipine, a dihydropyridine calcium channel blocker, could not block TWA or arrhythmias. Moreover, verapamil, but not nifedipine, significantly decreased ROS production in ischemic myocytes. Collectively, our results indicate that verapamil can significantly inhibit the development of cardiac alternans and ventricular arrhythmias in acute myocardial ischemia, and the mechanism was related to the inhibition of RyR and the protective function to oxidative stress.

Identification of Novel Reference Genes Suitable for qRT-PCR Normalization with Respect to the Zebrafish Developmental Stage.

Reference genes used in normalizing qRT-PCR data are critical for the accuracy of gene expression analysis. However, many traditional reference genes used in zebrafish early development are not appropriate because of their variable expression levels during embryogenesis. In the present study, we used our previous RNA-Seq dataset to identify novel reference genes suitable for gene expression analysis during zebrafish early developmental stages. We first selected 197 most stably expressed genes from an RNA-Seq dataset (29,291 genes in total), according to the ratio of their maximum to minimum RPKM values. Among the 197 genes, 4 genes with moderate expression levels and the least variation throughout 9 developmental stages were identified as candidate reference genes. Using four independent statistical algorithms (delta-CT, geNorm, BestKeeper and NormFinder), the stability of qRT-PCR expression of these candidates was then evaluated and compared to that of actb1 and actb2, two commonly used zebrafish reference genes. Stability rankings showed that two genes, namely mobk13 (mob4) and lsm12b, were more stable than actb1 and actb2 in most cases. To further test the suitability of mobk13 and lsm12b as novel reference genes, they were used to normalize three well-studied target genes. The results showed that mobk13 and lsm12b were more suitable than actb1 and actb2 with respect to zebrafish early development. We recommend mobk13 and lsm12b as new optimal reference genes for zebrafish qRT-PCR analysis during embryogenesis and early larval stages.

Ability of ambulatory ECG-based T-wave alternans to modify risk assessment of cardiac events: a systematic review.

BACKGROUND: Exercise-based spectral T-wave alternans (TWA) has been proposed as a noninvasive tool-identifying patients at risk of sudden cardiac death (SCD) and cardiac mortality. Prior studies have indicated that ambulatory electrocardiogram (AECG)-based TWA is an important alternative platform to exercise for risk stratification of cardiac events. This study sought to review data regarding 24-hour AECG-based TWA and to discuss its potential role in risk stratification of fatal cardiac events across a series of patient risk profiles. METHODS: Prospective clinical studies of the predictive value of AECG-based TWA obtained with daily activity published between January 1990 and November 2014 were retrieved. Major endpoints included composite endpoint of SCD, cardiac mortality, and severe arrhythmic events. RESULTS: Data were accumulated from 5 studies involving a total of 1,588 patients, including 317 positive and 1,271 negative TWA results. Compared with the negative group, positive group showed increased rates of SCD (hazard ratio [HR]: 7.49, 95% confidence interval [CI]: 2.65 to 21.15), cardiac mortality (HR: 4.75, 95% CI: 0.42 to 53.55), and composite endpoint (SCD, cardiac mortality, and severe arrhythmic events, HR: 5.94, 95% CI: 1.80 to 19.63). For the 4 studies evaluating TWA measured using the modified moving average method, the HR associated with a positive versus negative TWA result was 9.51 (95% CI: 4.99 to 18.11) for the composite endpoint. CONCLUSIONS: The positive group of AECG-based TWA has a nearly six-fold risk of severe outcomes compared with the negative group. Therefore, AECG-based TWA provides an accurate means of predicting fatal cardiac events.

Deep mRNA sequencing analysis to capture the transcriptome landscape of zebrafish embryos and larvae.

Transcriptome analysis is a powerful tool to obtain large amount genome-scale gene expression profiles. Despite its extensive usage to diverse biological problems in the last decade, transcriptomic researches approaching the zebrafish embryonic development have been very limited. Several recent studies have made great progress in this direction, yet the large gap still exists, especially regarding to the transcriptome dynamics of embryonic stages from early gastrulation onwards. Here, we present a comprehensive analysis about the transcriptomes of 9 different stages covering 7 major periods (cleavage, blastula, gastrula, segmentation, pharyngula, hatching and early larval stage) in zebrafish development, by recruiting the RNA-sequencing technology. We detected the expression for at least 24,065 genes in at least one of the 9 stages. We identified 16,130 genes that were significantly differentially expressed between stages and were subsequently classified into six clusters. Each revealed gene cluster had distinct expression patterns and characteristic functional pathways, providing a framework for the understanding of the developmental transcriptome dynamics. Over 4000 genes were identified as preferentially expressed in one of the stages, which could be of high relevance to stage-specific developmental and molecular events. Among the 68 transcription factor families active during development, most had enhanced average expression levels and thus might be crucial for embryogenesis, whereas the inactivation of the other families was likely required by the activation of the zygotic genome. We discussed our RNA-seq data together with previous findings about the Wnt signaling pathway and some other genes with known functions, to show how our data could be used to advance our understanding about these developmental functional elements. Our study provides ample information for further study about the molecular and cellular mechanisms underlying vertebrate development.

Clinical features of severe wasp sting patients with dominantly toxic reaction: analysis of 1091 cases.

BACKGROUND: Massive wasp stings have been greatly underestimated and have not been systematically studied. The aim of this study was to identify the clinical features and treatment strategies of severe wasp stings. METHODS AND FINDINGS: A multicenter retrospective study was undertaken in 35 hospitals and medical centers including 12 tertiary care hospitals and 23 secondary care hospitals in the Hubei Province, China. The detailed clinical data of 1091 hospitalized wasp sting patients were investigated. Over three-fourths (76.9%) of the cases had 10 or more stings and the in-hospital mortality of patients was 5.1%. Forty-eight patients died of organ injury following toxic reactions to the stings, whereas six died from anaphylactic shock. The in-hospital mortality in patients with >10 stings was higher than that of ≤10 stings (5.2% vs. 1.0%, p = 0.02). Acute kidney injury (AKI) was seen in 21.0% patients and most patients required blood purification therapy. Rhabdomyolysis was seen in 24.1% patients, hemolysis in 19.2% patients, liver injury in 30.1% patients, and coagulopathy in 22.5% patients. Regression analysis revealed that high creatinine level, shock, oliguria, and anemia were risk factors for death. Blood purification therapy was beneficial for patients with ≥20 stings and delayed hospital admission of patients (≥4 hours after sting). CONCLUSIONS: In China, most patients with multiple wasp stings presented with toxic reactions and multiple organ dysfunction caused by the venom rather than an anaphylactic reaction. AKI is the prominent clinical manifestation of wasp stings with toxic reaction. High creatinine levels, shock, oliguria, and anemia were risk factors for death.

[Progress on pluripotency factors in zebrafish].

The mammalian pluripotency factors, including transcription factors such as Pou5f1/Oct4, Sox2, Klf4 and Nanog, play critical roles in maintaining pluripotency of embryonic stem cells and inducing reprogramming of differentiated cells. However, the functions of vertebrate pluripotency factors in vivo have not been elucidated. Zebrafish(Danio rerio H.) is an excellent model for studying vertebrates' early embryo development. It allows functional studies of pluripotency factors to be conducted in an in vivo environment and therefore provides more accurate information on their roles. Nowadays, several homologs of mammalian pluripotency factors including oct4, nanog etc. have been identified in zebrafish. This review aimed at introducing the progress of the functional study on zebrafish pluripotency factors and comparing to those of other vertebrates.

NRSF/REST is required for gastrulation and neurogenesis during zebrafish development.

Repressor element 1-silencing transcription factor (REST) was recognized as a transcription suppressor regulating nervous system differentiation. However, the role of REST during early development has not been clarified. We cloned the zebrafish homolog of human REST. Real-time polymerase chain reaction results showed that zebrafish REST mRNA was both maternal and zygotic with the higher expression level from blastula to the late segmentation period. Whole-mount in situ hybridization showed that REST was strongly expressed in the blastoderm since dome stage and dynamically expressed mainly in developing brain, especially in the border of the brain subdivisions in early segmentation period. Knockdown of REST using translation blocking morpholino (MO-tra) technique resulted in gastrulation delay or even blockage, and subsequently led to embryo lethality by early segmentation period with deficient neurogenesis. However, splicing blocking morpholino for REST did not show obviously abnormal phenotype until 48 hpf (hours post-fertilization), indicating that maternal REST was an important regulator for gastrulation. Further study revealed that the abnormal development in MO-tra morphants was at least partly due to the dysfunction of protein transportation from the yolk to the blastoderm. Our results showed that REST (especially maternal supplied REST) was required for gastrulation and neurogenesis during zebrafish early embryogenesis.

Atoh8, a bHLH transcription factor, is required for the development of retina and skeletal muscle in zebrafish.

BACKGROUND: Math6/atoh8, a bHLH transcription factor, is thought to be indispensable for early embryonic development and likely has important roles in vertebrate tissue-specific differentiation. However, the function of Atoh8 during early development is not clear because homozygous knockout causes embryonic lethality in mice. We have examined the effects of the atoh8 gene on the differentiation of retina and skeletal muscle during early development in zebrafish. RESULTS: We isolated a Math6 homologue in zebrafish, designated as zebrafish atoh8. Whole -mount in situ hybridization analysis showed that zebrafish atoh8 is dynamically expressed mainly in developing retina and skeletal muscle. Atoh8-MO knock-down resulted in reduced eye size with disorganization of retinal lamination. The reduction of atoh8 function also affected the arrangement of paraxial cells and differentiated muscle fibers during somite morphogenesis. CONCLUSION: Our results show that Atoh8 is an important regulator for the development of both the retina and skeletal muscles necessary for neural retinal cell and myogenic differentiation during zebrafish embryogenesis.

U6 promoter-driven siRNA injection has nonspecific effects in zebrafish.

RNA interference (RNAi) is a posttranscriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA), which causes degradation of homologous mRNAs. RNAi has been observed in a wide range of eukaryotes, including fungi, plants and animals. In vertebrates, long dsRNA activates the interferon response and yields nonspecific degradation of mRNA. In contrast, small interference RNA (siRNA) duplexes with a length of 21-23 nucleotides trigger specific gene silencing and thus are widely used in gene function studies. The use of siRNA for gene silencing in zebrafish has rarely been reported. In this report, we studied mammalian U6 promoter-driven siRNA-mediated RNA interference in zebrafish. The well characterized genes Myf5, Dlg3 and Nacre were selected as targets. Two to four target siRNAs were synthesized with incorporation of the U6 promoter. Constructs were introduced into early zebrafish embryos through microinjection, followed by in situ hybridization and embryonic development was monitored to determine whether U6 promoter-driven siRNAs could efficiently suppress specific gene expression. We showed that these siRNAs could partially suppress endogenous gene expression and that the siRNA efficiency varied at different targeted positions. However, the U6 promoter-driven siRNAs may also have induced nonspecific gene suppression (off-target effects). It appears that, despite the findings of previous reports, the current methodology of siRNA interference is not practical for studying gene function during early zebrafish development.

Cloning and expression of translation elongation factor 2 (EF-2) in zebrafish.

We have identified a developmentally regulated gene translation elongation factor 2 (EF-2) in zebrafish (GenBank Accession No. AAQ91234). Analysis of DNA sequence of zebrafish EF-2 shows that the 2826 bp cDNA spans an open reading frame from nucleotide 55 to 2631 and encodes a protein of 858 amino acids. It shares an identity of 92, 93, 93, 92, 79 and 80% in amino acid sequence to human, mouse, Chinese hamster, Gallus gullus, C. elegans and Drosophila EF-2, respectively. Zebrafish EF-2 protein has 16 conserved domains, GTP-binding domain is found in the NH2 terminus, and the ADP-ribosylation domain locates at the COOH terminus. Whole mount in situ hybridization on zebrafish embryos shows that the transcripts of EF-2 gene are detected during the early development of zebrafish embryo and constantly change from 5-somite stage to protruding-mouth stage. It expresses strongly throughout envelope at 5-somite stage. Then the stained cells concentrate strongly in the eyes, brain and muscle tissue. From prim-25 stage the stained cells only appear strongly in the lens and the anterior portion of the cerebellum.

Expression and possible functional roles of cytochromes P450 2J1 (zfCyp 2J1) in zebrafish.

Cytochrome P450 2J (Cyp2J) subfamilies are recognized as catalysts of arachidonic acid metabolism in extrahepatic tissues of many species. However, to date, no P450 2J have been identified in zebrafish. We describe here a zfCyp2J1 cDNA which encodes a putative protein of 496 amino acids and shares 51%, 51%, 50%, 51% and 50% identity with mouse Cyp2J6, rabbit Cyp2J1, human Cyp2J2, cow Cyp2J2, and rat Cyp2J4, respectively. Despite detectable levels of expression by RT-PCR, no expression was shown by in situ hybridization using whole-mount tissues of the embryos. Gene-specific knockdown by antisense morpholino oligonucleotide had no phenotypic effect on embryonic development. However, over-expression of zfCyp2J1 by injection of the embryos with the cDNA resulted in substantial dose-dependent morphological defects. With adult zebrafish, whole-mount in situ hybridization showed that zfCyp2J1 was expressed predominantly in the brain and gonads. A semi-quantitative RT-PCR analysis further revealed that the zfCyp2J1 transcript was also expressed in the ovary, testis, heart, liver, and kidney. High levels of zfCyp2J1 mRNA were evident in primary growth stage (stage I) oocytes and cortical alveolus stage (stage II) oocytes but nearly undetectable in stage III and matured oocytes. These results suggest that zfCyp2J1 may not be involved in zebrafish embryogenesis but may rather play an important role in the functioning of brain and gonads of the adults. In addition, zfCyp2J1 may play a particularly crucial role in early oocyte maturation.

DAXX interacts with phage PhiC31 integrase and inhibits recombination.

Phage PhiC31 integrase has potential as a means of inserting therapeutic genes into specific sites in the human genome. However, the possible interactions between PhiC31 integrase and cellular proteins have never been investigated. Using pLexA-PhiC31 integrase as bait, we screened a pB42AD-human fetal brain cDNA library for potential interacting cellular proteins. Among 61 positives isolated from 10(6) independent clones, 51 contained DAXX C-terminal fragments. The strong interaction between DAXX and PhiC31 was further confirmed by co-immunoprecipitation. Deletion analysis revealed that the fas-binding domain of DAXX is also the region for PhiC31 binding. Hybridization between a PhiC31 integrase peptide array and an HEK293 cell extract revealed that a tetramer, 451RFGK454, in the C-terminus of PhiC31 is responsible for the interaction with DAXX. This tetramer is also necessary for PhiC31 integrase activity as removal of this tetramer resulted in a complete loss of integrase activity. Co-expression of DAXX with PhiC31 integrase in a HEK293-derived PhiC31 integrase activity reporter cell line significantly reduced the PhiC31-mediated recombination rate. Knocking down DAXX with a DAXX-specific duplex RNA resulted in increased recombination efficiency. Therefore, endogenous DAXX may interact with PhiC31 causing a mild inhibition in the integration efficiency. This is the first time that PhiC31 was shown to interact with an important cellular protein and the potential effect of this interaction should be further studied.

Cloning and developmental expression of the DEC1 ortholog gene in zebrafish.

DEC1/STRA13/SHARP2 is a transcription factor of the bHLH family that has been suggested to play key roles in mammalian cell differentiation, the cell cycle and circadian regulation. However, the function of the DEC1 gene during embryogenesis is not well understood. In the present study, we cloned a zebrafish ortholog of the human DEC1 gene and analyzed its expression during development of zebrafish. The predicted protein encoded by zebrafish DEC1 consists of 403 amino acids, and shares 59%, 60% and 59% identity in overall amino acid sequence with human DEC1, mouse STRA13 and rat SHARP2, respectively. Zebrafish DEC1 contains a bHLH domain exhibiting 97% identity with that of the mammalian ortholog. During zebrafish embryogenesis, DEC1 is expressed in a strong ubiquitous manner before early segmentation. At 15-72 hpf, DEC1 shows a specific and dynamic expression in the developing eyes, somites, pineal gland (epiphysis), heart, brain, spinal cord, notochord, pronephric duct, common cardinal vein and blood cells. In older zebrafish, DEC1 also is expressed in multiple tissues including the brain, eye, gut, liver and pancreas. Our data provide evidence that expression of DEC1 is evolutionally conserved in zebrafish.

Cloning, expression and functional study of translation elongation factor 2 (EF-2) in zebrafish.

We have identified translation elongation factor 2 (EF-2) in zebrafish (GenBank Accession No. AAQ91234). Analysis of the DNA sequence of zebrafish EF-2 shows that the 2826 bp cDNA spans an open reading frame between nucleotide 55 to 2631 and encodes a protein of 858 amino acids. Zebrafish EF-2 protein shares 92%, 93%, 93% and 92% identity with the corresponding amino acid sequence in human, mouse, Chinese hamster and Gallus EF-2, respectively. Whole-mount in situ hybridization showed that zebrafish EF-2 was a developmentally regulated gene and might play important roles during the early development of zebrafish embryos. Therefore, we further studied the function of EF-2 during early embryogenesis. Using morpholino antisense oligo knockdown assays, anti-MO injected embryos were found to display abnormal development. The yolk balls were larger than normal and the melanophores spreading on their bodies became fewer. Furthermore, their tails were incurvate and their lenses were much smaller than those of the normal embryos. However the EF-2 overexpression data showed that extra EF-2 protein had no obvious effect on zebrafish embryonic development.

Slit-like 2, a novel zebrafish slit homologue that might involve in zebrafish central neural and vascular morphogenesis.

Nervous and vascular systems grow as parallel networks, indicating common cues in distal targets. We have identified a novel zebrafish gene slit-like 2 (slitl2) that might involve in zebrafish central neural and vascular morphogenesis. Whole-mount in situ hybridization of zebrafish embryo detected distinct signals of slitl2 transcripts in zebrafish midline structure of central nervous system similar to that of slits. Strong expression is also observed in zebrafish vasculature. Zebrafish slitl2 shares amino acid sequence identity of 41% with Homo sapiens slitl2 (vasorin) and Mus musculus slitl2, and 35%, 33% with Danio rerio slit3, slit2. Analysis of zebrafish slitl2 cripto growth factor domain, extracellular matrix protein slit domain, and putative signal peptide confirms that as a secreted and cell-surface protein slitl2 may be essential in axon guidance, vessel development, and axis patterning. These results provide evidence that slitl2 may play important roles in zebrafish central nervous system and vascular morphogenesis.

Molecular cloning and expression of two closely related GTP-binding proteins from zebrafish.

Developmentally regulated GTP-binding proteins (DRGs) are a subclass of GTP-binding proteins that have been discovered recently. Here we report two zebrafish DRG cDNA clones closely related to human and mouse DRG genes. The two DRG sequences showed a high degree of similarity (55% identity, 72% similarity) at the amino acids level. Whole mount in situ hybridization revealed expression of zebrafish DRGs maternally, following the onset of zygotic transcription at the mid-blastula transition (MBT) and throughout embryonic. The expression of these two genes in different tissues follows a similar pattern, suggesting that they may serve a similar function.

Enhancement of naked FIX minigene expression by chloroquine in mice.

AIM: To study the effect of chloroquine on the expression of human clotting factor IX (hFIX) in mice. METHODS: Hydrodynamics-based naked DNA plasmid administration was performed by tail vein injection of 10 microg of pCMV- hFIX and chloroquine (0, 100, 200, and 500 micromol/L) in 2.2 mL of Ringer's solution within 6-7 s, the level and stability of hFIX expression, liver damage and toxicity were then examined. RESULTS: The maximum expression of hFIX level was 4.4+/-1.8 mg/L at 8 h after injection, 9.7+/-1.6 mg/L at 24 h only existed in 200 micromol/L chloroquine-treated animals, which is 3-4 fold higher than that of control (P<0.01). There is no significant difference observed among all the treated groups, 3 d later. Transaminase level and liver histological study showed the damage of liver was not related to chloroquine (P>0.05). CONCLUSION: Chloroquine can enhance and sustain exogenous gene expression in vivo without side effect under our experimental conditions.