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Abstrak :
Principles of Cloning, Second Edition is the fully revised edition of the authoritative book on the science of cloning. The book presents the basic biological mechanisms of how cloning works and progresses to discuss current and potential applications in basic biology, agriculture, biotechnology, and medicine. Beginning with the history and theory behind cloning, the book goes on to examine methods of micromanipulation, nuclear transfer, genetic modification, and pregnancy and neonatal care of cloned animals. The cloning of various species-including mice, sheep, cattle, and non-mammals-is considered as well. The Editors have been involved in a number of breakthroughs using cloning technique, including the first demonstration that cloning works in differentiated cells done by the Recipient of the 2012 Nobel Prize for Physiology or Medicine - Dr John Gurdon; the cloning of the first mammal from a somatic cell - Drs Keith Campbell and Ian Wilmut; the demonstration that cloning can reset the biological clock - Drs Michael West and Robert Lanza; the demonstration that a terminally differentiated cell can give rise to a whole new individual - Dr Rudolf Jaenisch and the cloning of the first transgenic bovine from a differentiated cell - Dr Jose Cibelli. The majority of the contributing authors are the principal investigators on each of the animal species cloned to date and are expertly qualified to present the state-of-the-art information in their respective areas. First and most comprehensive book on animal cloning, 100% revisedDescribes an in-depth analysis of current limitations of the technology and research areas to exploreOffers cloning applications on basic biology, agriculture, biotechnology, and medicine.

Abstrak :
Describes the miraculous breakthroughs scientists have made in the past decade and dispels the many myths and misunderstandings surrounding stem cell research and therapeutic cloning, offering a window into the astonishing potential of regenerative medicine to extend the human life span and cure disease. Annotation. The extraordinary story of the breakthrough discoveries in cell aging, stem cell research, and therapeutic cloning, and the tremendous promise they hold for dramatically extending human life. Dr. Michael West has been consumed with the mystery of science since he was as an inquisitive child mixing chemicals in his attic-turned-laboratory. Today, he stands in the center of a controversy so great that the list of those lining up against him includes President George W. Bush. Once a devoted creationist eager to dispel theories of human evolution, Dr. West was set on a quest to find a scientific solution to the devastating effects of disease and death after the death of his father. He became immersed in the study of cell aging and the discovery of the cellular "clock" telomerase - the mechanism that controls cell aging. His work led him to found the biotechnology company Geron, a pioneer in the field of stem cell research. His new company, Advanced Cell Technology, is the only organization in the United States pursuing human therapeutic cloning research - research in the field of "regenerative medicine" intended to repair damaged and diseased human organs and tissues. Unlike reproductive cloning, the attempt to clone a human child, therapeutic cloning is a process of growing cells, using a patient's own DNA that is inserted it into an unfertilized egg cell to create embryonic stem cells, cells that hold the promise of repairing the damage of age and disease - in essence, making the cell young again. The potential for therapeutic cloning to treat afflictions caused by the loss of dysfunction of cells - from spinal cord injury and skin burns to kidney failures and cancer - is enormous. Part memoir, part adventure story, The Immortal Cell chronicles the breakthroughs Dr. West and other scientists have made in biotechnology over the past decade - and the astonishing potential they offer us to cure diseases and improve the quality of human life.

Abstrak :
Penelitian bertujuan memperoleh clone gen NS1 DEN-3 strain CH53489 pada vektor ekspresi pET-21d(+) agar gen tersebut dapat diekspresikan oleh Escherichia coli (Migula) Castellani & Chalmers. Penelitian dilakukan di Laboratorium Biopharming, Balai Pengkajian dan Penerapan Teknologi (BPPT), Serpong selama 12 bulan (Maret 2006--Maret 2007). Gen NS1 dengue berukuran 1.050 pb diperbanyak dengan polymerase chain reaction (PCR) menggunakan pasangan primer spesifik (NS1-F dan NS1-R). Vektor ekspresi pET-21d(+) (berukuran 5.440 pb) diperbanyak melalui teknik cloning dalam E. coli DH5α. Produk PCR dan vektor ekspresi pET-21d(+) didigesti dengan BamHI dan XhoI, kemudian diligasi secara in vitro dan ditransformasi dengan kejutan panas ke dalam sel kompeten E.coli DH5α. Sebanyak 129 koloni diperoleh pada medium seleksi SOB ampisilin agar. Sebanyak 26 dari 129 koloni kandidat rekombinan dipilih secara acak untuk diverifikasi menggunakan enzim restriksi (BamHI, XhoI, dan NcoI) dan selanjutnya dilakukan PCR. Verifikasi menghasilkan 3 koloni positif rekombinan. Clone 6D pembawa gen NS1 dengue diverifikasi lebih lanjut melalui sequencing dengan primer T7 promoter. Hasil sequencing dibandingkan dengan data referensi dalam database DNA GenBank melalui pencarian homologi BLASTN. Hasil BLAST menunjukkan sekuens dari clone gen NS1 dengue memiliki homologi sebesar 99% dengan sekuens DEN-3 strain FW06 no. AY858041.1. Clone gen NS1 dengue dalam vektor ekspresi pET-21d(+) telah berhasil diperoleh, namun dengan tingkat keberhasilan yang rendah.

Abstrak :
Contents : V. 1. Pt. 1. Essentials -- V. 2. Pt. 2. Analysis and manipulation of DNA and RNA ; Pt. 3. Introducing genes into cells -- V. 3. Pt. 4. Gene expression ; Pt. 5. Interaction Analysis ; Appendices.

Abstrak :
ABSTRACT
Dengue is an infectious disease caused by dengue virus. Dengue endemic region includes America, Western Pacific, Africa, East Mediterranian, and South East Asia including Indonesia. An early diagnostic system specific for Indonesia is needed to control dengue in Indonesia. In this research, cloning of Non Structural 1 (NS1) gene from dengue virus type 3 (Indonesian strain D3-1703) into pYES2/CT vector was performed. In the long run, NS1 recombinant protein will be expressed in Saccharomyces cerevisiae for diagnostic materials. Polymerase Chain Reaction (PCR) amplification of NS1 gene fragments were done with optimal annealing temperature at 55 ºC. NS1 gene fragment and pYES2/CT were cut by Bam H I and Not I enzymes. The digested pYES2/CT was dephosphosrylated using Calf Intestine Alkaline Phospatase enzyme. Ligation with the vector:insert ratio of 1:12 and 1:20 resulted in 6 and 5 recombinant colony candidates respectively. Restriction enzyme and PCR verifications showed that 5 recombinant plasmids contained NS1 gene. Sequencing of the first 600 bp of one recombinant plasmid was performed. The blastn analysis showed that it had a 99% identity with dengue virus type 3 strain FW06. Finally, it was shown that NS1 clone within pYES2/CT was in the correct Open Reading Frame and ready to be expressed in S. cerevisiae.

Abstrak :
Spag11a diketahui terekspresi secara spesifik pada kaput epididimis sehingga dimungkinkan protein tersebut memiliki fungsi yang spesifik untuk maturasi spermatozoa. Studi peran SPAG11A dalam maturasi spermatozoa di epididimis memerlukan produksi protein SPAG11A untuk dikarakterisasi. Tujuan dari penelitian ini adalah untuk mengklon, mengekspreesikan, dan mengkarakterisasi sifat antimikroba dari protein rekombinan SPAG11A. Insert cDNA Spag11a yang dihasilkan melalui PCR diklon ke dalam vektor pET100/D-TOPO. Plasmid rekombinan kemudian diekspresikan ke Escherichia coli BL21 DE3 star. Deteksi dari fusi protein rekombinan dilakukan dengan SDS-PAGE dan Western Blotting. IMAC Immobilized Metal Affinity Chromatography digunakan untuk mempurifikasi protein rekombinan. Uji antimikroba protein rekombinan dianalisis melalui pengukuran Optical density.PCR amplifikasi dari cDNA kaput epididimis mencit menghasilkan insert Spag11a berukuran 210bp. Insert tersebut kemudian dikloning ke dalam pET100/D-TOPO menghasilkan 1 rekombinan plasmid dari 10 koloni yang diskrining. Ekspresi rekombinan klon ke dalam E.coli BL21 menghasilkan fusi protein setelah diinduksi IPTG selama 4 jam. Fusi protein dikonfirmasi menggunakan Western Blotting menggunakan antibodi yang mengenali N-terminal His-Tag 21kDa dan protein SPAG11A. Uji antimikroba protein rekombinan SPAG11A mununjukkan tidak ada inhibisi yang signifikan terhadap laju pertumbuhan E.coli dan Bacillus subtilis. Insert Spag11a yang berukuran 210bp berhasil diklon ke dalam vektor pET100/D-TOPO. Ekspresi rekombinan Spag11a menghasilkan fusi protein berukuran 21kDa. Protein rekombinan SPAG11A tidak membawa sifat antimikroba terhadap E.coli dan B. subtilis. ...... Spag11a is known to be specifically expressed in the caput region of the epididymis suggesting a specific function for sperm maturation. Study of SPAG11A role in the epididymal sperm maturation requires generating SPAG11A protein for characterization. The objective of this study was to clone, express and characterize antimicrobial property of the recombinant SPAG11A. Spag11a cDNA insert was generated by PCR and cloned in TOPO vector. Recombinant DNA plasmid was subsequently expressed in E coli BL 21 star. Detection of recombinant fusion protein was carried out using SDS PAGE and western immunobloting. IMAC Immobilized Metal Affinity Chromatography was used to purify recombinant protein. Optical density measurement was used to analyse antimicrobial property of the recombinant protein. PCR amplification of mouse caput epididymis cDNA produced a 210 bp insert of Spag11a. Cloning of the insert into TOPO pET100 resulted in 2 recombinants out of 10 colonies that were screened. Expression of recombinant clones in the E coli BL21 produced a fusion protein after being induced IPTG for 4 hours. Fusion protein was confirmed by western immunobloting using two antibodies recognizing N terminal His Tag 21 kDa and SPAG11A protein. Antimicrobial assay for SPAG11A recombinant showed no significant inhibition towards growth rates of E coli and Bacillus subtilis. A 210 bp Spag11a insert was successfully cloned into TOPO pET100 vector. Expression of recombinant spag11a produced a fusion protein of 21 kDa. SPAG11A recombinant protein does not have antimicrobial property towards E coli and B subtilis.

Abstrak :
Latar belakang: Protein VP6 rotavirus adalah protrein struktural utama yang berperan penting selama replikasi, dan merupakan bagian yang paling lestari dan memiliki potensi dalam menstimulasi respon imun, yaitu sebagai protein target yang dapat menstimulasi sel T, dan memiliki epitop yang cross-reactive di antara genotipe rotavirus lainnya, sehingga memiliki potensi untuk dikembangkan sebagai vaksin. Tujuan: Untuk mengetahui karakterisasi molekular protein VP6 rotavirus strain Indonesia, serta pengklonaan gen dan ekspresi protein VP6 pada Escherichia coli BL21 untuk pengembangan vaksin rotavirus. Metode: RNA rotavirus R55 dan R10 diperoleh dari ekstraksi sampel klinis. RNA tersebut kemudian diamplifikasi dengan reaksi RT-PCR dan menghasilkan amplikon 1194 bp yang selanjutnya disekuensing untuk konfirmasi dan mengetahui karakterisasi molekular protein VP6 secara bioinformatika. Gen VP6 sebagai sisipan dan plasmid pQE-80L sebagai vektor direstriksi ganda dengan enzim restriksi dan diligasi menggunakan enzim ligasi. Produk ligasi ditransformasikan pada sel kompeten E.coli Top 10 dan diseleksi klon pembawa plasmid rekombinan. Plasmid rekombinan yang mengandung gen VP6 ditransformasikan ke sel kompeten E.coli BL21. Ekspresi protein dilakukan dengan induksi IPTG. Hasil ekspresi dianalisis dengan SDS-PAGE dan dikonfirmasi dengan Uji Western Blot. Hasil: Sekuen asam amino gen VP6 rotavirus strain Indonesia R55 dan R10 memiliki tingkat homologi yang tinggi dengan epitop yang lestari bila dibandingkan dengan strain vaksin dan kandidat vaksin rotavirus; strain R55 lebih dekat kekerabatannya dengan rotavirus genotipe I, strain R10 lebih dekat kekerabatannya dengan rotavirus genotipe II. Sekuen VP6 rotavirus strain R55 dan R10 menunjukkan tingkat hidrofobisitas yang sama, hal ini mengindikasikan sejenis protein permukaan atau protein yang bersifat hidrofilik. Hasil analisa struktur sekunder pada strain R55 dan R10 menunjukkan adanya perbedaan pada posisi asam amino 149-152 dan 341-349. Telah didapatkan klon pQE-80L yang mengandung gen VP6 dari strain R55 dan R10. Ekspresi protein VP6 pada SDS-PAGE menunjukkan adanya perbedaan intensitas pita protein antara sel E. coli yang diinduksi IPTG dengan yang tidak diinduksi, mengindikasikan protein VP6 diduga berhasil diekspresikan. Konfirmasi ekspresi protein menggunakan Western-Blot menunjukkan hasil yang sama antara sel yang diinduksi dengan yang tidak diinduksi, namun hasil ini perlu dikonfirmasi lebih lanjut. Kesimpulan: Hasil karakterisasi molekular protein VP6 rekombinan dan pengklonaan gen VP6 dari rotavirus strain Indonesia R55 dan R10 dapat dikembangkan sebagai studi awal pada pengembangan vaksin subunit berbasis protein VP6 rekombinan. Namun untuk tahap ekspresi protein rekombinan VP6 perlu optimasi lebih lanjut. Latar belakang: Protein VP6 rotavirus adalah protrein struktural utama yang berperan penting selama replikasi, dan merupakan bagian yang paling lestari dan memiliki potensi dalam menstimulasi respon imun, yaitu sebagai protein target yang dapat menstimulasi sel T, dan memiliki epitop yang cross-reactive di antara genotipe rotavirus lainnya, sehingga memiliki potensi untuk dikembangkan sebagai vaksin. Tujuan: Untuk mengetahui karakterisasi molekular protein VP6 rotavirus strain Indonesia, serta pengklonaan gen dan ekspresi protein VP6 pada Escherichia coli BL21 untuk pengembangan vaksin rotavirus. Metode: RNA rotavirus R55 dan R10 diperoleh dari ekstraksi sampel klinis. RNA tersebut kemudian diamplifikasi dengan reaksi RT-PCR dan menghasilkan amplikon 1194 bp yang selanjutnya disekuensing untuk konfirmasi dan mengetahui karakterisasi molekular protein VP6 secara bioinformatika. Gen VP6 sebagai sisipan dan plasmid pQE-80L sebagai vektor direstriksi ganda dengan enzim restriksi dan diligasi menggunakan enzim ligasi. Produk ligasi ditransformasikan pada sel kompeten E.coli Top 10 dan diseleksi klon pembawa plasmid rekombinan. Plasmid rekombinan yang mengandung gen VP6 ditransformasikan ke sel kompeten E.coli BL21. Ekspresi protein dilakukan dengan induksi IPTG. Hasil ekspresi dianalisis dengan SDS-PAGE dan dikonfirmasi dengan Uji Western Blot. Hasil: Sekuen asam amino gen VP6 rotavirus strain Indonesia R55 dan R10 memiliki tingkat homologi yang tinggi dengan epitop yang lestari bila dibandingkan dengan strain vaksin dan kandidat vaksin rotavirus; strain R55 lebih dekat kekerabatannya dengan rotavirus genotipe I, strain R10 lebih dekat kekerabatannya dengan rotavirus genotipe II. Sekuen VP6 rotavirus strain R55 dan R10 menunjukkan tingkat hidrofobisitas yang sama, hal ini mengindikasikan sejenis protein permukaan atau protein yang bersifat hidrofilik. Hasil analisa struktur sekunder pada strain R55 dan R10 menunjukkan adanya perbedaan pada posisi asam amino 149-152 dan 341-349. Telah didapatkan klon pQE-80L yang mengandung gen VP6 dari strain R55 dan R10. Ekspresi protein VP6 pada SDS-PAGE menunjukkan adanya perbedaan intensitas pita protein antara sel E. coli yang diinduksi IPTG dengan yang tidak diinduksi, mengindikasikan protein VP6 diduga berhasil diekspresikan. Konfirmasi ekspresi protein menggunakan Western-Blot menunjukkan hasil yang sama antara sel yang diinduksi dengan yang tidak diinduksi, namun hasil ini perlu dikonfirmasi lebih lanjut. Kesimpulan: Hasil karakterisasi molekular protein VP6 rekombinan dan pengklonaan gen VP6 dari rotavirus strain Indonesia R55 dan R10 dapat dikembangkan sebagai studi awal pada pengembangan vaksin subunit berbasis protein VP6 rekombinan. Namun untuk tahap ekspresi protein rekombinan VP6 perlu optimasi lebih lanjut. ......Background: VP6 protein is an intermediate layer on rotavirus outer capsid shell which is the major structural protein and play an important role in replication cycle. VP6 protein is a conserved region that can induce immune response as target protein of T cell with cross-reactive epitopes within another genotypes. Objectives: This research conducted to determine the molecular characterization of rotavirus VP6 recombinant protein of Indonesia strain, and to determine the clone and expression of VP6 protein in Escherichia coli BL21 for development of rotavirus vaccine. Methods: Rotavirus RNA was extracted from clinical sample of R55 and R10 strains that having correlation with genotipes I and II rotavirus. RNA samples were amplified with RT-PCR reaction and produced 1194 bp amplicon, and sequencing reaction were conducted to confirm and analyze the molecular characterization of VP6 protein in bioinformatics. VP6 gene as insert and pQE-80L plasmids as vector were double restricted and then ligated by ligation enzyme. Product of ligation were transformed to E.coli Top 10 competent cells and the clones were selected to get the recombinant plasmids which bearing VP6 gene. The recombinant plasmid than subcloned to E.coli BL21 competent cells and induced by IPTG and its pellets were loaded directly onto SDS-PAGE, approximately 45 kDa protein was observed on the SDS-PAGE. The protein was analyzed using Western-blotting. Results: Level of amino acids homology from R55 and R10 rotavirus strain compared with vaccine strains and vaccine candidate strains showed high level of homology, with the conserved regions of T-cell epitopes. R55 strain having close relativity with genotype I while R10 strain having close relativity with genotype II. there are the same level of hydrophobicity between R55 and R10 which indicated as surface proteins or as a hydrophilic protein. The differences of secondary structure between R55 and R10 in amino acids position of 149-152 and 341-349. The VP6 cloned obtained from E.coli Top 10 with pQE-80L plasmid. The profile of expressed VP6 recombinant protein from R55 and R10 strains in SDS-PAGE showed the different intensity of protein between induced condition with IPTG and non-induced condition, indicated that VP6 protein might be successfully expressed. The Western-Blot assay showed the same result between the cell that induced and non-induced, but it still need another confirmation. Conclusion: The result of molecular characterization from VP6 recombinant protein and the cloned of VP6 gene that obtained from R55 and R10 rotavirus strains of Indonesia could be applied as a preliminary study to develop rotavirus candidate vaccine based on subunit vaccine.

Abstrak :
Protein PD-1 biasanya diekspresikan berlebihan pada pasien kanker dan menghambat sistem imun. Antibodi monoklonal dari PD-1 dapat digunakan untuk menghambat jaras tersebut. Namun, urutan nukelotida dari epitop dengan afinitas yang kuat masih belum diketahui. Oleh karena itu, plasmid yang mengandung epitop kecil dari PD-1 dibuat untuk proses epitope mapping. Tujuan penelitian ini adalah untuk mendapatkan plasmid yang mengandung daerah N-terminal dari gen PD-1. DNA insert sintetik dibuat dari metode PCR dan dipurifikasi. Kedua DNA insert dan plasmid pQE80L didigesti dengan enzim restriksi BamH1 dan HindIII, dipurifikasi dan diligasi. Plasmid tersebut dimasukkan kedalam sel kompeten TOP10 dengan transformasi heat shock. Koloni positif diseleksi menggunakan metode PCR koloni dan verifikasi dengan menggunakan digesti dan sanger sequencing. Produk PCR dari EP1PD1 didapat dengan menggunakan suhu annealing sebesar 57ºC dan berhasil diligasi ke plasmid pQE80L and ditransformasikan. Hasil dari sequencing menunjukan urutan yang sama namun terdapat insersi diawal. Beberapa modifikasi perlu dilakukan untuk mengekspresi protein EP1PD1. Konklusi dari penelitian ini adalah plasmid yang mengandung epitop 1 PD-1 berhasil diperoleh dengan insersi. ......PD-1 protein tends to be overexpressed in cancer patient and inhibits immune system. Monoclonal antibody of PD-1 can be used to inhibit this pathway. However, the sequence of epitope with strong affinity is currently unknown. Thus, plasmid containing small epitope of PD-1 was made for PD-1 epitope mapping process. The objective of this research is to obtain plasmid containing N-terminal region of PD-1 gene. Synthetic insert DNA was made using PCR method and purified. Both insert DNA and pQE80L plasmid were digested using BamH1 and HindIII enzyme, purified and ligated. It was then inserted into TOP10 competent cell using heat shock transformation method. Positive colonies are selected using PCR colony and verified using digestion and Sanger sequencing. PCR product of EP1PD1 are obtained using annealing temperature of 57ºC and able to be ligated to pQE80L plasmid and transformed. Sequencing result shows EP1PD1 result with insertion in the beginning. Modifications are required to express EP1PD1 protein. In conclusion, the plasmid containing Epitope 1 of PD-1 are able to be obtained with insertion.