Generating pAAV-U6-mCherry Construct

To generate pAAV-U6-mCherry, the mCherry sequence was PCR amplified from pAAV-Ef1a-DIO-DSE-mCherry-PSE-shRNA. AvrII-WPRE-pA (purchased from Addgene; Cat #129669), the EGFP sequence was eliminated from pAAV-U6-EGFP by digestion with MluI and BsrGI, and the mCherry sequence was subcloned into the vector’s MluI/BsrGI enzyme site. The following plasmids were obtained commercially: rAAV2-retro helper (Addgene; Cat# 81070), pAAV-RAM-d2TTA::TRE-NLS-mKate2-WPREpA (Addgene; Cat# 84474), pAAV-hSynI-FLEX-TVA-P2A-EGFP-2A-oG (Addgene; Cat# 85225), EnvA G-deleted Rabies-mCherry (Addgene; Cat# 32626), and pAAV-EF1α-fDIO-hM4Di-mCherry (Addgene; Cat# 50461). pAAV-EF1α-fDIO-NLS-GFP-∆Cre and pAAV-EF1α-fDIO-NLS-GFP-Cre were constructed by PCR amplification of the NLS-∆Cre-GFP and NLS-Cre-GFP segment from pAAV-hSynI-NLS-∆Cre-GFP and pAAV-hSynI-NLS-Cre-GFP vector, respectively, followed by subcloning into the pAAV-EF1α-fDIO at BsrGI and NheI sites. The following plasmids were previously described: pAAV-U6-EGFP^{80 (link)}; pAAV-hSynI-∆Cre-EGFP and pAAV-hSynI-Cre-EGFP^{42 (link)} and pAAV-phSynI-WGA-IRES-mCherry-Flpo-bGHGpA^{81 (link)}; and pAAV-DIO-hM3Dq-2A-mCherry^{82 (link)}.

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Han K.A., Yoon T.H., Kim J., Lee J., Lee J.Y., Jang G., Um J.W., Kim J.K, & Ko J. (2024). Specification of neural circuit architecture shaped by context-dependent patterned LAR-RPTP microexons. Nature Communications, 15, 1624.

Publication 2024

rAAV2-retro helper

Corresponding Organization :

Other organizations : Daegu Gyeongbuk Institute of Science and Technology, Korea Basic Science Institute, Pohang University of Science and Technology

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Variable analysis

independent variables

The mCherry sequence was PCR amplified from pAAV-Ef1a-DIO-DSE-mCherry-PSE-shRNA.
The EGFP sequence was eliminated from pAAV-U6-EGFP by digestion with MluI and BsrGI.
The mCherry sequence was subcloned into the vector's MluI/BsrGI enzyme site.

dependent variables

Not explicitly mentioned.

control variables

Not explicitly mentioned.

controls

Positive control: pAAV-U6-EGFP (previously described).
Negative control: Not explicitly mentioned.

Annotations

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