BaculovirusMediated Expression in Mammalian Cells'

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Baculovirus-Mediated Expression in Mammalian
Cells.
5 / 27 / 2003 Jong-Chan, AHN
Interdisciplinary Graduate Program in Genetic
Engineering (IGPGE)
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Enhancement and Prolongation of
Baculovirus-Mediated Expression in Mammalian
Cells Focuses on Strategic Infection and Feeding
Yu-Chen Hu, Chien-Tai Tsai, Yao-Jen Chang, and
Jen-Huang Huang Department of Chemical
Engineering, National Tsing Hua University,
Hsinchu, Taiwan 300
Biotechnol. Prog. 2003, 19, 373-379

• The baculovirus/insect cell system has been
  widely used for recombinant protein
  production.
• 2. In this study, They intended to explore
  the possibility of utilizing a baculovirus/mammali
  an cell system.
• 3. A recombinant baculovirus vector carrying
  EGFP under the control of CMV-IE promoter was
  constructed.
• 4. HeLa was found to yield the highest
  expression level.

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How did they treat it?
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Construction of the plasmid pBac-CMV-EGFP
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The egfp gene along with the upstream CMV-IE
promoter was amplified by PCR from pEGFP-C1
plasmid (Clontech) using the following primers

• 5-CGCG AGATCT TAG TTA TTA ATA GTA ATC AAT TA-3
• 5-AAGCTT TTA CTT GTA CAG CTC GTC CAT GCC G-3
• (enzyme sites Bgl II and Hind III are
  underlined).

Where?
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pEGFP-C1
CGCG AGATCT
Bgl II
AA AAGCTT
Hind III
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The PCR product was inserted into the BamH I/Hind
III sites of pFastBac DUAL (Invitrogen, Carlsbad,
CA), which placed the expression cassette in
multiple cloning site (MCS) I under the
polyhedrin promoter. The resultant plasmid was
designated pBac-CMV-EGFP as shown before. This
arrangement placed both polyhedrin and CMV-IE
promoter in tandem and upstream of the egfp gene
and conferred the EGFP expression in both insect
and mammalian cells.
BamH I
Bgl II
5'-AG A T C T-3 3'-T C T A GA-5'
5'-GG A T C C-3 3'-C C T A GG-5'
The polh-CMV-IE-EGFP expression cassette was
transferred from pBac-CMV-EGFP to a bacmid in
DH10Bac E. coli by site-specific transposition.
pFASTBAC DUAL allows for the cloning and
simultaneous expression of two heterologous
proteins.
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RESULTS

• Bright-field (upper panel) and fluorescence
  (lower panel) micrographs of four cell lines
  (HeLa, Cos7, BHK, and CHO) infected by Bac-CE (24
  hpi).
• The infection was conducted in 12 well
  plates at MOI 200 when cells were at 1 x 105
  cells/well.
• Variation of the percentage of GFP cells
  infected by Bac-CE with time.
• The percentage was measured by
  comparison of fluorescing and nonfluorescing
  cells.

The virus infection did not result in apparent
cell death or growth arrest hence, the cells
continued to divide, albeit at a slightly slower
rate (compared with uninfected cells, not shown).
Moreover, the virus does not naturally replicate
in the mammalian cells (15) thus, the cell
division resulted in a decrease in the percentage
of GFP cells after 24 hpi.
NONETHELESS
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It was observed that a significant portion of the
new daughter cells also emitted fluorescence
after 72 h , which suggested that the baculovirus
genomes were distributed to the new cells during
the cell division cycle.
Time course profiles of the total FI in four cell
lines infected by Bac-CE.
Fluorescing intensity (FI) of individual
daughter cells
HeLa cells yielded the highest overall EGFP
expression that culminated at 96 hpi.
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How can we use it better ?
Is it possible to make a good mammalian
expression system ?
So
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Lets make a
Baculo-Tet off Tet on
Multiple Epitope tagging
mammalian Expression Vector !!!
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Modified Baculo Donor plasmid ? ??
First, Amplify. (Use PCR)
And then,
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Second, Remove Polh prometer at pFastBac1.
Third, Cloning TRE CMV promoter region at this
position.
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Is it OK?
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Fourth, Amplify Red BOX.
Defective enhancer elements !
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Defective enhancer elements