Wednesday, June 22, 2011 9:13:54 AM
Bacillovirus insertion in silkworms has no significance for KBLB
Firstly it's over 5 years old, which pretty much attests to its irrelevance.
Secondly it uses a vector for insertion which, like all others except for zinc fingers, is random. So the gene, although expressed, was highly unlikely to have been expressed in the right tissue and under the right circumstances. It was not even tested to see where and when it was expressed nor was there any indication whatsoever that the silk was even tested.
Bottom line: completely irrelevant.
Note that the experiment was not intended to affect the production of silk (which explains the lack of testing to see where and when the gene was expressed (but only to see whether it was expressed at all) and lack of testing of silk. It was clearly only intended to determine if the vector (baculovirus) used for genetic modification was capable of inserting genes into silkworms. It was. But only randomly which is why in the 6 years since, it hasn't been used to put any spider genes into silkworms at sites where they would actually affect silk production.
[[Note also that it refers to "traditional homologous recombination method" which I pointed out before was a dead standard method and NOT a propietary method as claimed by Entogenetics.]]
http://onlinelibrary.wiley.com/doi/10.1111/j.1439-0418.2006.01051.x/abstract
Silkworm, Bombyx mori larvae expressed the spider silk protein through a novel Bac-to-Bac/BmNPV baculovirus
Y.-G. Miao1,2, A.-C. Zhao2, Y.-S. Zhang2, K. Nakagaki2, Y. Meng2, T.-F. Zhao2, M. Nakagaki2
Article first published online: 22 MAY 2006
DOI: 10.1111/j.1439-0418.2006.01051.x
Issue
Journal of Applied Entomology
Volume 130, Issue 5, pages 297–301, June 2006Keywords:
Bombyx mori;
Bac-to-Bac/BmNPV baculovirus expression system;
silkworm larvae;
spider flagelliform silk protein (Flag)
Abstract: The silkworm has become an ideal multicellular eukaryotic model system for basic research. The major advantages of expressing foreign genes in silkworm larvae are the low cost of feeding, the extremely high levels of expression achievable compared with expression in cell lines and increased safety because the baculovirus is noninfectious to vertebrates. In this study, we used a recently developed Bombyx mori Nucleopolyhedrovirus (BmNPV) bacmid to express the spider flagelliform silk gene in silkworm larvae. The recombinant bacmid baculoviruses (rBacmid/BmNPV/Flag) were introduced into the first-day larvae of the fifth instar by subcutaneous injection. The worms presented symptoms typical of NPV infection from 72 h after injection compared with control. The haemolymph was collected from the infected larvae 120 h post-infection and the recombinant 6× His-tagged Flag protein was purified by the Ni-NTA spin kit under denaturing conditions with 8 m urea. A 37.0-kDa protein was visualized both in rBacmid/BmNPV/Flag-infected haemolymph and eluting fraction. The results showed that the Bac-to-Bac/BmNPV baculovirus expression system is an efficient tool to express the target gene in silkworm larvae, which takes only 7–10 days for generating recombinant baculovirus, compared with the traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses
Firstly it's over 5 years old, which pretty much attests to its irrelevance.
Secondly it uses a vector for insertion which, like all others except for zinc fingers, is random. So the gene, although expressed, was highly unlikely to have been expressed in the right tissue and under the right circumstances. It was not even tested to see where and when it was expressed nor was there any indication whatsoever that the silk was even tested.
Bottom line: completely irrelevant.
Note that the experiment was not intended to affect the production of silk (which explains the lack of testing to see where and when the gene was expressed (but only to see whether it was expressed at all) and lack of testing of silk. It was clearly only intended to determine if the vector (baculovirus) used for genetic modification was capable of inserting genes into silkworms. It was. But only randomly which is why in the 6 years since, it hasn't been used to put any spider genes into silkworms at sites where they would actually affect silk production.
[[Note also that it refers to "traditional homologous recombination method" which I pointed out before was a dead standard method and NOT a propietary method as claimed by Entogenetics.]]
http://onlinelibrary.wiley.com/doi/10.1111/j.1439-0418.2006.01051.x/abstract
Silkworm, Bombyx mori larvae expressed the spider silk protein through a novel Bac-to-Bac/BmNPV baculovirus
Y.-G. Miao1,2, A.-C. Zhao2, Y.-S. Zhang2, K. Nakagaki2, Y. Meng2, T.-F. Zhao2, M. Nakagaki2
Article first published online: 22 MAY 2006
DOI: 10.1111/j.1439-0418.2006.01051.x
Issue
Journal of Applied Entomology
Volume 130, Issue 5, pages 297–301, June 2006Keywords:
Bombyx mori;
Bac-to-Bac/BmNPV baculovirus expression system;
silkworm larvae;
spider flagelliform silk protein (Flag)
Abstract: The silkworm has become an ideal multicellular eukaryotic model system for basic research. The major advantages of expressing foreign genes in silkworm larvae are the low cost of feeding, the extremely high levels of expression achievable compared with expression in cell lines and increased safety because the baculovirus is noninfectious to vertebrates. In this study, we used a recently developed Bombyx mori Nucleopolyhedrovirus (BmNPV) bacmid to express the spider flagelliform silk gene in silkworm larvae. The recombinant bacmid baculoviruses (rBacmid/BmNPV/Flag) were introduced into the first-day larvae of the fifth instar by subcutaneous injection. The worms presented symptoms typical of NPV infection from 72 h after injection compared with control. The haemolymph was collected from the infected larvae 120 h post-infection and the recombinant 6× His-tagged Flag protein was purified by the Ni-NTA spin kit under denaturing conditions with 8 m urea. A 37.0-kDa protein was visualized both in rBacmid/BmNPV/Flag-infected haemolymph and eluting fraction. The results showed that the Bac-to-Bac/BmNPV baculovirus expression system is an efficient tool to express the target gene in silkworm larvae, which takes only 7–10 days for generating recombinant baculovirus, compared with the traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses
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