KONSTRUKSI VEKTOR EKSPRESI GEN HORMON PERTUMBUHAN LELE DUMBO (Clarias sp.) UNTUK PRODUKSI IKAN LELE LOKAL (Clarias batrachus) TRANSGENIK

Penelitian ini bertujuan untuk memperoleh konstruksi vektor ekspresi (pTarget) rekombinan yang mengandung sisipan gen hormon pertumbuhan ikan lele dumbo (Clarias sp.) dan promoter β-aktin ikan lele lokal (C. batrachus) dalam upaya pembuatan ikan lele lokal transgenik. Promoter β-aktin lele lokal (pCbBA) telah berhasil diisolasi dari hipofisa ikan tersebut dengan ukuran sekitar 1,7 kbp; dan memiliki elemen transkripsi CAAT box, TATA box, GC box, motif CarG, dan TGACG berdasar analisis program TF Bind. Penggantian promoter CMV (cytomegalovirus) yang terkandung dalam vektor ekspresi pTarget menggunakan dua enzim restriksi SgfI dan I-PpoI, menghasilkan fragmen DNA berukuran 6.083 bp (pTarget-GH lele dumbo) sebagai produk digesti. Fragmen pTarget-GH lele dumbo yang diligasi dengan promoter β-aktin lele lokal membentuk konstruksi By: Ibnu Dwi Buwono, Nono Carsono, Yuniar Mulyani, and M. Untung KurniaThis study aims to obtain an expression vector construct (pTarget) containing recombinant growth hormone gene insertion of African catfish (Clarias sp.) and β-actin promoter derived from walking catfish (Clarias batrachus) in order to produce transgenic local catfish. β-actin promoter of walking catfish (pCbBA) have been isolated from the pituitary of the fish with a size of about 1.7 kbp, and has a transcription element: CAAT box, TATA box, GC box, CarG, and TGACG motif based on analysis result of TF Bind program. Replacement of CMV (cytomegalovirus) promoter contained in the expression vector pTarget using restriction enzymes SgfI and I-PpoI, obtained a product of digestion with the fragment size of 6,083 bp (pTarget-GH African catfish). pTarget-GH fragments were ligated with the African catfish β-actin promoter to arrange a construct of pTarget-pCbBA-African catfish GH (7,783 bp) as transgenic walking catfish expression vector.

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ISOLATION OF Β-ACTIN PROMOTER DERIVED FROM WALKING CATFISH (CLARIAS BATRACHUS)

KnE Life Sciences ◽

10.18502/kls.v1i0.105 ◽

2015 ◽

Vol 2 (1) ◽

pp. 201

Author(s):

Ibnu Dwi Buwono ◽

Nono Carsono ◽

Yuniar Mulyani ◽

Mochamad Untung Kurnia Agung

Keyword(s):

Growth Hormone ◽

Expression Vector ◽

Fish Growth ◽

Clarias Batrachus ◽

Growth Hormone Gene ◽

Economic Traits ◽

Food Material ◽

Vector Construction ◽

Reverse Primer ◽

Actin Promoter

Fish growth improvement as economic traits can be solved through fish transgenic production. Growth hormone gene is inserted into transgenic vector construction to over-express fish growth. The promoter as a part of the expression vector has an important role in its regulation. The use of promoter which is derived from mammalian or virus (such as CMV/ cytomegalovirus) in the expression vector, in specific goal as food material, has customer resistant rather than a promoter which is derived from in- sibling species. Beside of it, transgene expression level when using in-sibling promoter showed higher than using mammalian or viral promoter. The β-actin promoter is screened from walking catfish pituitary genome DNA using primers: pBA-cy-F (5’- GTGWGTGACGCYGGACCAAATC-3’) as forward primer and pBA-cy-R (5’- CCATRTCRTCCCAGTTGGTSACAAT-3’) as reverse primer, produced an amplicon of 1,7 kb in length. Sequence analysis using TF BindTM indicated transcription factor elements: TATA box, CCAAT box, enhancer (CAAT), and CarGG (CAAATGG) motif. This result showed that promoter which is obtained from this research is useful in construction of all catfish growth hormone vector expression catfish transgenic production. Keywords: β-actin promoter, growth hormone, expression vector, walking catfish (Clarias batrachus)

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Characterization of LL25X, a Newly Isolated Anti-SIV Monoclonal Antibody, to Determine Binding Specificity

Elements ◽

10.6017/eurj.v13i1.9604 ◽

2017 ◽

Vol 13 (1) ◽

Author(s):

Zackary Tajin Park

Keyword(s):

Phage Display ◽

Mammalian Cells ◽

Expression Vector ◽

Restriction Enzymes ◽

Phage Display Library ◽

Single Chain ◽

Phage Display Technology ◽

Mammalian Expression ◽

Mammalian Expression Vector ◽

Single Chain Fv

A phage display library was previously constructed from an SIV-infected rhesus macaque. Several single chain Fv (scFv), including SU24, SU343 and LL25X, were selected using phage display technology. Sequences corresponding to SU24, SU343 and LL25X were optimized for expression in a mammalian system and commercially synthesized. SU24 and SU343 had previously been cloned into a mammalian expression vector. In this study, we aimed to characterize the specificity of SU24, SU343, and LL25X.. The codon-optimized version of the scFv LL25X gene sequence was cloned into a mammalian expression vector (pCEP4). LL25X DNA was amplified by PCR, and the PCR product and mammalian expression vector were both digested with KpnI/SapI restriction enzymes. Digested fragments were purified, and the fragments were ligated using T4DNA ligase. E. coli cells were transformed with the ligation reaction. Single colonies were selected on LB agar plates containing the selective antibiotic (ampicillin). Positive colonies were identified after DNA mini-preparation and test-digestion with KpnI and SapI. Sanger sequencing confirmed cloning results and DNA sequence accuracy. Following transfection of mammalian cells (293T), LL25X-Fc cells, and purifying our protein, the binding of LL25X-Fc to the SIV gp140 envelope protein was confirmed via ELISA and Western Blotting.

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Regulation of protamine gene expression in an in vitro homologous system.

Acta Biochimica Polonica ◽

10.18388/abp.1996_4506 ◽

1996 ◽

Vol 43 (2) ◽

pp. 369-377 ◽

Cited By ~ 1

Author(s):

J M Jankowski ◽

P D Cannon ◽

F Van der Hoorn ◽

L D Wasilewska ◽

N C Wong ◽

...

Keyword(s):

Gene Expression ◽

Regulatory Elements ◽

In Vitro Transcription ◽

Tata Box ◽

Transcription System ◽

Proposed Model ◽

Binding Factor ◽

Gc Box ◽

Responsive Element

An in vitro transcription system from the trout testis nuclei was developed to study trout protamine gene expression. The protamine promoter contains, among others, two regulatory elements: 1) a cAMP-responsive element or CRE element (TGACGTCA) which is present in position 5' to TATA box, and 2) GC box (CCGCCC) which is present in position 3' to TATA box. The removal of the CRE-binding protein by titration (by the addition of appropriate oligonucleotides to the incubation mixture) resulted in a decrease in transcription of the protamine gene. These results were confirmed by experiments in which the pure CRE-binding factor (TPBP1) was used, as well as by those where a stimulatory effect of cAMP on protamine promoter transcription was observed. On the other hand, addition of oligonucleotides containing the GC-box sequence enhanced the protamine gene transcription indicating that the protein (Sp1 like) which binds to this sequence acts as a repressor of protamine gene expression. These results confirm the previously proposed model which suggested that the GC box played a role in negative regulation of the protamine gene expression. Involvement of some other factors in this process was also discussed.

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Cloning and sequencing of a grass carp growth hormone gene and construction of an all-fish recombinant growth hormone gene

Aquaculture ◽

10.1016/0044-8486(93)90075-a ◽

1993 ◽

Vol 111 (1-4) ◽

pp. 308

Author(s):

Zhu Zuoyan ◽

He Ling ◽

Xie Yuefeng ◽

Lo Guohua

Keyword(s):

Growth Hormone ◽

Grass Carp ◽

Growth Hormone Gene ◽

Cloning And Sequencing ◽

Recombinant Growth Hormone

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Construction of Expression Vector with Fruit-Specific Promoter and Genetic Transformation of Strawberry

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.690-693.1314 ◽

2013 ◽

Vol 690-693 ◽

pp. 1314-1317

Author(s):

Xiang Li Song ◽

Zhi Hui Wu ◽

Xi Zhang ◽

Meng Xi Lu ◽

Bei Guo

Keyword(s):

Expression Vector ◽

Staining Method ◽

Tomato Fruit ◽

Expression System ◽

Restriction Enzymes ◽

Histochemical Staining ◽

Sequencing Analysis ◽

Gene Encoding ◽

Plant Expression Vector ◽

E8 Promoter

In order to transcription and expression an exogenous gene just in transgenic strawberry fruit, the gene encoding tomato fruit-specific E8 promoter was cloned. We constructed plant expression vector with E8 promoter replace the CaMV35S of pBI121 that used for transformation of strawberry. Recombination plasmid was identified by PCR, restriction enzymes digestion and sequencing analysis, and transfered into the agrobacterium EHA105 strain. At the same time, the agrobacterium EHA105, transformation and no transformation tissue of strawberry used as the experiment materials to study the expression of reporter genegusAby the histochemical staining method. The results show that agrobacterium which containsgusAgene and strawberry organization of transformation can be dyed blue, others were not stain. So in testing the transgenic plants by agroinfiltration transient expression system can reduce or avoid false positive appearance.

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The GC box as a silencer

Bioscience Reports ◽

10.1007/bf01122129 ◽

1987 ◽

Vol 7 (12) ◽

pp. 955-963 ◽

Cited By ~ 11

Author(s):

Jacek M. Jankowski ◽

Gordon H. Dixon

Keyword(s):

Rainbow Trout ◽

Binding Site ◽

Tata Box ◽

Sperm Cells ◽

Promoter Regions ◽

Protein Factor ◽

Eukaryotic Genes ◽

Initiation Of Transcription ◽

Multiple Copies ◽

Gc Box

A DNA control sequenceTGGGGCGGAATGGC, or the “GC” box, has been described in the promoter regions upstream of a number of eukaryotic genes transcribed by polymerase II (for review, see Dynan, W. S. and Tjian, R., Nature316:774, 1985). The “GC” box can occur in single or multiple copies and is the binding site for a protein factor, Spl, which activates initiation of transcription. We have observed in the rainbow trout protamine gene 3′ to the TATA box, three “GC” boxes spaced at 80 bp intervals. The first is 5′ to the cap site and possesses the ability to “silence” transcription from the protamine promoter in constructs linking this promoter to the bacterial chloramphenicol acetyl transferase (CAT) coding sequence following transfection to COS-1 cells. A model is proposed to account for the silencing of the protamine gene in all tissues except developing sperm cells.

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DISTRIBUTION AND EXPRESSION OF STRIPED CATFISH (Pangasionodon hypophtalmus) GROWTH HORMONE GENE (PhGH) IN THE ORGAN OF AFRICAN CATFISH (Clarias gariepinus) TRANSGENIC FOUNDER

Indonesian Aquaculture Journal ◽

10.15578/iaj.7.1.2012.11-18 ◽

2012 ◽

Vol 7 (1) ◽

pp. 11

Author(s):

Huria Marnis ◽

Raden Roro Sri Pudji Sinarni Dewi ◽

Imron Imron ◽

Bambang Iswanto

Keyword(s):

Growth Hormone ◽

Clarias Gariepinus ◽

Stomach Muscle ◽

African Catfish ◽

Growth Hormone Gene ◽

Caudal Fin ◽

Expression Levels ◽

Striped Catfish ◽

Pcr Method ◽

High Level

Faster growing African catfish can be produced by transgenesis. This study was conducted to investigate the distribution and expression of growth hormone gene (PhGH) in various organs of the transgenic African catfish (Clarias gariepinus) founder (F0). Transgene was detected using the PCR method in various organs, namely pituitary, brain, liver, heart, spleen, kidney, intestine, stomach, muscle, caudal fin, gill and eye. Transgene expression levels were analyzed using the method of reverse transcriptase-polymerase chain reaction (RT-PCR), -actin gene used as internal controls. The results showed that the PhGH was detected and expressed in all organs of the transgenic African catfish founder. The high level of PhGH expression was found in the liver, pituitary, intestine and brain; smaller amounts were detectable in muscle, spleen, kidneys, heart, and stomach, caudal fin, gill and eyes, range from 0.02-0.75 PhGH/-actin mRNA. The expression levels of PhGH had positive correlation with tissue and body size (P<0.05).

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Study of indigenous freshwater fish diversity of Bankura (West Bengal), India with special reference to Clarias batrachus

Journal of Applied and Natural Science ◽

10.31018/jans.v10i4.1892 ◽

2018 ◽

Vol 10 (4) ◽

pp. 1162-1172

Author(s):

Arindam Ganguly ◽

Amrita Banerjee ◽

Asish Mandal ◽

Tapas Kumar Dutta ◽

Pradeep Kumar Das Mohapatra

Keyword(s):

Freshwater Fish ◽

Fish Species ◽

West Bengal ◽

Clarias Batrachus ◽

Fish Diversity ◽

African Catfish ◽

Hypophthalmichthys Molitrix ◽

Local Markets ◽

Wallago Attu ◽

Bankura District

A study was conducted to determine the ichthyofaunal diversity in Bankura district of West Bengal. The area is traversed by major and minor rivers, feeder channels, numerous ponds, bills, reservoirs which have made this drought-trodden district as the highest producer of aquatic products within the state. Field survey was conducted in randomly selected local markets covering twenty-two blocks of Bankura. Fish sampling was carried out using various conventional fishing gears. The entire region harbours a wide variety of aquatic fishes. A total number of 92 indigenous fish species belonging to 30 families were identified during this study. The Cyprinidae family dominated the population with its 36 varieties followed by Channidae, Siluridae and Bagridae. Sonamukhi block situated in Shali basin; Sarenga and Raipur of Kangsabati basin; Dwarkeshwar and seven bundhs- enriched Bishnupur were the major habitats of small indigenous fishes. This paper also denotes presence of 12 globally endemic freshwater fish species viz. Hypophthalmichthys molitrix, Cyprinus carpio, Labeo nandina, Tor khudree, Chitala chitala, Bagarius bagarius, Wallago attu, Ompok pabda, Ailia coila, Anguilla bengalensis, Parambassis lala, Oreochromis mossambicus. The substitution of native Clarias batrachus with invasive African catfish C. gariepinus was observed in various local markets. Some traders even promote illicit farming of this banned species for their own profit. As a consequence, the indigenous, nutritionally-enriched C. batrachus is becoming more endangered.

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