Jenn-Kan Lu

Suppression of myostatin with vector-based RNA interference causes a double-muscle effect in transgenic zebrafish.

Myostatin belongs to the transforming growth factor (TGF)-beta superfamily and is a potent negative regulator of skeletal muscle development and growth. We utilized microinjection of an antisense RNA-expressing vector to establish a hereditarily stable myostatin gene knockdown zebrafish strain with a double-muscle phenotype. Real-time PCR and immunostaining revealed that the myostatin messenger (m)RNA and protein levels in homozygous transgenic zebrafish were 33% and 26% those of the non-transgenic controls, respectively. Also, the mRNA levels of myogenic regulatory factor markers such as MyoD, myogenin, Mrf4, and Myf5 were dramatically elevated in myostatin-suppressed transgenic fish compared to the non-transgenic controls. Although there was no significant difference in body length, homozygous transgenic zebrafish were 45% heavier than non-transgenic controls. Histochemical analysis showed that the cross-sectional area of the muscle fiber of homozygous transgenic fish was twice as large as that of non-transgenic controls. This is the first model zebrafish with a hereditarily stable myostatin-suppressed genotype and a double-muscle phenotype.

Production of Transgenic Silver Sea Bream (Sparus sarba) by Different Gene Transfer Methods

We have been interested in developing convenient mass gene transfer methods for producing strains of silver sea bream (Sparus sarba) with superior genetic traits for aquaculture. A transgene construct carrying rainbow trout growth hormone (rtGH) complementary DNA driven by a common carp b-actin promoter was introduced into silver sea bream by electroporating the sperm with the rtGH transgene and using the treated sperm to fertilize eggs stripped from mature females. The presence of the GH transgene in presumptive transgenic individuals was detected by polymerase chain reaction (PCR) analysis. Between 56% and 70% of the animals carried the GH transgene. We refer to this method as sperm-mediated gene transfer (SMGT). Since the handling stress of stripping gametes from female sliver sea bream brood fish could cause severe mortality, an alternative gene transfer method would be highly desirable. We developed a liposome-based method to transfer the GH transgene into the fish. This method, referred as testis-mediated gene transfer (TMGT), involves injecting the liposome-transgene mixture into the gonads of male sea bream at least 48 hours before spawning. The males were mated to reproductively active females, and fertilized eggs were collected for further incubation. Between 59% and 76% of the hatched fry were found by PCR analysis to carry the rtGH transgene. The efficiency of gene transfer was improved more than 80% by injecting multiple doses of the liposome-transgene mixture into the gonads of treated males. Results of Southern blot analysis of DNA isolated from PCR-positive animals showed that the transgene was integrated into the host genome and could be transmitted to its offspring. The rtGH transgene was expressed in many of the rtGH-transgenic fish. Several P1 GH-transgenic silver sea bream exhibited significant growth enhancement compared with nontransgenic controls. Our studies showed that faster-growing silver sea bream could be produced by a variety of mass gene transfer technologies. These gene transfer technologies would be of great value to aquaculture.

Molecular Cloning and Characterization of cDNA of Penaeidin-like Antimicrobial Peptide from Tiger Shrimp (Penaeus monodon)

We report the molecular cloning and characterization of a penaeidin-like antimicrobial peptide (AMP) complementary DNA from the hemocytes of tiger shrimp (Penaeus monodoni). A tiger shrimp AMP cDNA containing the entire coding region of the peptide, determined by rapid amplification of cDNA 5′ and 3′ ends and polymerase chain reaction of the messenger RNA isolated from hemocytes of the shrimp, is 683 bp in length with an open reading frame of 222 bp. The deduced amino acid sequence of this antimicrobial peptide consists of 55 amino acid residues of the mature peptide and a signal peptide of 19 amino acid residues. The mature peptide contains a proline-rich domain at the N terminus and 6 cysteine residues at the C terminus, and it shares less than 50% amino acid sequence identity with the mature penaeidins of Litopenaeus vannamei. To demonstrate the bactericidal activity of this peptide, a synthetic peptide was prepared according to the amino acid sequence deduced from the cDNA of tiger shrimp penaeidin-like AMP. By the minimal inhibitory concentration MIC assay, the synthetic peptide was shown to exert bactericidal activity against Escherichia coli, Vibrio harveyi, Vibrio alginolyticu, and Aerococcus viridans. It also inhibited the growth of 2 filamentous fungi, Fusarium pisi and Fusarium oxysporum. Tiger shrimp penaeidin-like AMP mRNA was detected in hemocytes, gills, intestines, eyestalks, hepatopancreas, and muscles of the tiger shrimp by reverse transcriptase–polymerase chain reaction assay. Although the highest level was detected in hemocytes, challenging tiger shrimp with V. harveyi did not result in a significant increase of the mRNA level in hemocytes.

Molecular Characterization of Tetralogy of Fallot Within DiGeorge Critical Region of the Chromosome 22

Abstract. The purpose of this study was to determine whether the levels of heterozygosity and microdeletion of specific loci within the DiGeorge critical region (del22q11) are associated with different phenotypes of tetralogy of Fallot (TF). Examinations were conducted on 84 sporadic TF patients and their unaffected parents for del22q11, using the following 9 simple tandem repeat polymorphic microsatellite markers: D22S420, D22S427, D22S941, D22S944, D22S264, D22S311, D22S425, D22S303, D22S257. The microdeletions were confirmed using quantitative PCR with markers TUPLE1, exon 2 of the UFD1L gene, and D22S264; the boundaries of these microdeletions were estimated using genotypic analyses of the unaffected family members. The del22q11 was identified in 14 patients (16.6%). The boundary of the shortest region of deletion overlap (SRO) in these 14 TF patients was identified, proximally using D22S427 and distally using the TUPLE 1 gene. The deletion of exon 2 of the UFD1L gene and TUPLE1 gene was identified in 13 patients (13/14 cases; 93%). The SRO in TF patients with del22q11 was at or close to the ADU breakpoint and centromeric to the UFD1L gene. The level of heterozygosity for the marker D22S944 in TF patients without del22q11 (n= 70) was found to be significantly lower than expected. Overall, this study demonstrated the significantly low level of heterozygosity within DiGeorge critical region in TF patients with or without del22q11. Our results suggest that the genetic factors leading to DiGeorge/velocardiofacial syndrome might also be partly responsible for TF phenotypes.

Induction of apoptosis and inhibition of cell growth by tbx5 knockdown contribute to dysmorphogenesis in Zebrafish embryos

BackgroundThe tbx5 mutation in human causes Holt-Oram syndrome, an autosomal dominant condition characterized by a familial history of congenital heart defects and preaxial radial upper-limb defects. We report aberrant apoptosis and dormant cell growth over head, heart, trunk, fin, and tail of zebrafish embryos with tbx5 deficiency correspond to the dysmorphogenesis of tbx5 morphants.MethodsWild-type zebrafish embryos at the 1-cell stage were injected with 4.3 nl of 19.4 ng of tbx5 morpholino or mismatch-tbx5-MO respectively in tbx5 morphants and mismatched control group. Semi-quantitative RT-PCR was used to for expression analysis of apoptosis and cell cycle-related genes. TUNEL and immunohistochemical assay showed the apoptosis spots within the local tissues. Ultra-structure of cardiac myocardium was examined by transmission electron microscope.ResultsApoptosis-related genes (bad, bax, and bcl2), and cell cycle-related genes (cdk2, pcna, p27, and p57) showed remarkable increases in transcriptional level by RT-PCR. Using a TUNEL and immnuohistochemical assay, apoptosis was observed in the organs including the head, heart, pectoral fins, trunk, and tail of tbx5 knockdown embryos. Under transmission electron microscopic examination, mitochondria in cardiomyocytes became swollen and the myocardium was largely disorganized with a disarrayed appearance, compatible with reduced enhancement of myosin in the cardiac wall. The ATP level was reduced, and the ADP/ATP ratio as an apoptotic index significantly increased in the tbx5 deficient embryos.ConclusionOur study highlighted that tbx5 deficiency evoked apoptosis, distributed on multiple organs corresponding to dysmorphogenesis with the shortage of promising maturation, in tbx5 knockdown zebrafish embryos. We hypothesized that mesenchymal cell apoptosis associated with altered TBX5 level may subsequently interfered with organogenesis and contributed to dysmorphogenesis in tbx5 deficiency zebrafish embryos.

Marine Microbial Biosurfactin

The marine biosphere offers a wealth of flora and fauna and represents a vast natural resource of imperative functional commercial-grade products. Marine bacterial natural products are predicted to become an important resource for drug discovery and enzyme mining. Among the various bioactive compounds, biosurfactants (BS ) are attracting major interest and attention due to their structural and functional diversity. New techniques for the isolation of these marine bacteria are opening the possibility for the isolation of novel and potential products and processes from them. This will also help to improve our understanding of marine bacterial physiology, genetics, biochemistry, and ecology in order to provide model systems for research and production systems for commerce, and to contribute to understanding and conservation of the seas. The recent discovery of new secondary metabolites from novel bacteria and new approaches for accessing gene products from marine bacteria are contributing to the increasing development of the field. Here, we discuss the role and applications of marine microbial biosurfactants, focusing on applications for cosmeceutical and nutraceutical industry perspectives. Nutraceuticals are food products that provide health as well as medical benefits, including the prevention and treatment of disease. Few nutraceuticals are being used as pharmaceuticals and a number of others are being used and purchased by the general public as self-medication. This article outlines biosurfactants as a novel nutraceutical with their therapeutic and cosmeceutical applications.

Anti-apoptotic effects of IGF-I on mortality and dysmorphogenesis in tbx5-deficient zebrafish embryos

BackgroundTbx5 deficiency in zebrafish causes several abnormal phenotypes of the heart and pectoral fins. It has been reported that exogenous human growth hormone can enhance expression of downstream mediators in the growth hormone and insulin-like growth factor I (IGF-I) pathway and partially restore dysmorphogenesis in tbx5 morphants. This study aimed to further evaluate the effects of IGF-I on cell apoptosis and dysmorphogenesis in zebrafish embryos deficient for tbx5.ResultsAmong the five studied groups of zebrafish embryos (wild-type embryos [WT], tbx5 morphants [MO], mismatched tbx5 morpholino-treated wild-type embryos [MIS], IGF-I-treated wild-type embryos [WTIGF1], and IGF-I-treated tbx5 morphants [MOIGF1]), the expression levels of the ifg1, igf1-ra, ifg-rb, erk1, and akt2 genes as well as the ERK and AKT proteins were significantly reduced in the MO group, but were partially restored in the MOIGF1 group. These expression levels remained normal in the WT, MIS, and WTIGF1 groups. Exogenous human IGF-I also reduced the incidence of phenotypic anomalies, decreased the expression levels of apoptotic genes and proteins, suppressed cell apoptosis, and improved survival of the MOIGF1 group.ConclusionsThese results suggest that IGF-I has an anti-apoptotic protective effect in zebrafish embryos with tbx5 deficiency.

Complete Genome Sequence of Streptococcus iniae 89353, a Virulent Strain Isolated from Diseased Tilapia in Taiwan

ABSTRACT Streptococcus iniae 89353 is a virulent strain isolated from diseased tilapia in Taiwan. The full-genome sequence of S. iniae 89353 is 2,098,647 bp. The revealed genome information will be beneficial for identification and understanding of potential virulence genes of Streptococcus iniae and possible immunogens for vaccine development against streptococcosis.