Part:BBa_K5353061

1364 LentiV-MscS/HA: EGFP/Neo

After purify the plasmid DNA through midi-prep, we utilize them to package the lentivirus together with DVPR and VSV-G. We utilize these lentivirus to infect HEK293T cell and the successfully infected cells will express the MscS-HA tag protein. Meanwhile, this plasmid will also express green fluorescent protein (GFP). The MscS could be induced by physical stimulation (such as ultrasound) to induce the calcium overload. The HA tag could be used for Western blot and imaging detection of the MscS protein, while GFP can be used for validation of the lentivirus infection. The lentivirus gene sequence could be integrated into HEK293T cell genome by utilizing G418 selection (by the Neo marker gene).

The plasmid information.

1364-linear

1364-plasmid

The designed DNA was constructed and we used the midi-prep preparation kit to conduct the plasmid isolation and purification of the plasmid DNA (Table 1). The inserts in each construct were further validated by the PCR reaction (Figure 1. and Figure 2).

Table 1.DNA concentrations extracted and purified.

Figure 1. Gel electrophoresis results 1.

Figure 2. Gel electrophoresis results 2.

The successful infection could be visualized through fluorescence microscopy.(Figure 1364-1.)

Figure 1364-1.Expression of green fluorescent protein of the 1833 HEK293T cell.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2227
  Illegal XhoI site found at 2626
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2361
  Illegal AgeI site found at 1739
  
• 1000
  COMPATIBLE WITH RFC[1000]