Difference between revisions of "Part:BBa K3652006"

Revision as of 07:59, 26 October 2020

Lentiviral Vector shRNA Regulatory Region (pLKO-tet-neo)

This part is the H1 promoter, tet-on operator system, and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). It can be the regulatory precursor for any shRNA sequence inserted into a generation 3 lentiviral cloning (transfer) plasmid, such as the tet-pLKO-neo plasmid which we inserted this part into. The human H1 promoter is a Pol III promoter commonly used for RNAi applications--specifically, as the promoter for siRNA or shRNA coding sequences. It is slightly weaker than the additionally common promoter, U6, with typically lower expression levels and shorter-term effects when compared on similar genes and shRNA sequences [1]. We used it preceding our shRNA sequence, because we wanted to avoid irreversible gene knockdown, or very long-term effects in our target users, as it could lead to excess angiogenesis, and harm the patients. Ultimately H1 is a good choice for those looking for a weaker knockdown than that typically seen with U6 promoters. The tet-on operator system is a very common regulatory system, developed in 1995 by Gossen et. al. It allows the activation of gene expression in the presence of tetracycline, or doxycycline via a variant of the TetR protein (developed by mutagenesis) [2]. This variant binds to tetO in the presence of tet/dox. Finally, the EMCV IRES was used as the viral ribosome binding site. Though not strictly necessary for successful transcription of the following shRNA/siRNA sequence, it has been shown to increase efficacy of expression of the shRNA [3], so we included it seeking more successful transcription of the shRNA, at least during the testing. If it validates the transformation of our plasmid, and produces high amounts of shRNA, its removable can be considered to alter the potency of the shRNA production.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 66
Illegal PstI site found at 274
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 274
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 66
Illegal PstI site found at 274
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 66
Illegal PstI site found at 274
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3652006 short</partinfo>
-This composite part is the H1 promoter, tet-on operator system, and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). It can be a precursor for any shRNA sequence inserted into a generation 3 lentiviral cloning (transfer) plasmid, such as the tet-pLKO-neo plasmid which we inserted this part into.  The human H1 promoter is a Pol III promoter commonly used for RNAi applications--specifically, as the promoter for siRNA or shRNA coding sequences. It is slightly weaker than the additionally common promoter, U6, with typically lower expression levels and shorter-term effects when compared on similar genes and shRNA sequences [1]. We used it preceding our shRNA sequence, because we wanted to avoid irreversible gene knockdown, or very long-term effects in our target users, as it could lead to excess angiogenesis, and harm the patients. Ultimately H1 is a good choice for those looking for a weaker knockdown than that typically seen with U6 promoters. The tet-on operator system is a very common regulatory system, developed in 1995 by Gossen et. al. It allows the activation of gene expression in the presence of tetracycline, or doxycycline via a variant of the TetR protein (developed by mutagenesis) [2]. This variant binds to tetO in the presence of tet/dox. Finally, the EMCV IRES was used as the viral ribosome binding site. Though not strictly necessary for successful transcription of the following shRNA/siRNA sequence, it has been shown to increase efficacy of expression of the shRNA [3], so we included it seeking more successful transcription of the shRNA, at least during the testing. If it validates the transformation of our plasmid, and produces high amounts of shRNA, its removable can be considered to alter the potency of the shRNA production.
+This part is the H1 promoter, tet-on operator system, and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). It can be the regulatory precursor for any shRNA sequence inserted into a generation 3 lentiviral cloning (transfer) plasmid, such as the tet-pLKO-neo plasmid which we inserted this part into.  The human H1 promoter is a Pol III promoter commonly used for RNAi applications--specifically, as the promoter for siRNA or shRNA coding sequences. It is slightly weaker than the additionally common promoter, U6, with typically lower expression levels and shorter-term effects when compared on similar genes and shRNA sequences [1]. We used it preceding our shRNA sequence, because we wanted to avoid irreversible gene knockdown, or very long-term effects in our target users, as it could lead to excess angiogenesis, and harm the patients. Ultimately H1 is a good choice for those looking for a weaker knockdown than that typically seen with U6 promoters. The tet-on operator system is a very common regulatory system, developed in 1995 by Gossen et. al. It allows the activation of gene expression in the presence of tetracycline, or doxycycline via a variant of the TetR protein (developed by mutagenesis) [2]. This variant binds to tetO in the presence of tet/dox. Finally, the EMCV IRES was used as the viral ribosome binding site. Though not strictly necessary for successful transcription of the following shRNA/siRNA sequence, it has been shown to increase efficacy of expression of the shRNA [3], so we included it seeking more successful transcription of the shRNA, at least during the testing. If it validates the transformation of our plasmid, and produces high amounts of shRNA, its removable can be considered to alter the potency of the shRNA production.
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