Transcript Cloning of TfRGFP in Sindbis

Cloning of TfRGFP in Sindbis
Ashley Angell*, Begum Choudhury, and Dr. Dax A. Hoffman, Ph.D.
Molecular Neurophysiology and Biophysics Unit, Laboratory of Cellular and Synaptic Neurophysiology,
Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA
INTRODUCTION
Gene Kv4.2 encodes a voltage-gated A-type potassium
ion channel protein, which belongs to the delayed rectifier class
and Sha1 potassium channel subfamily. Voltage-gated potassium
ion channels (Kv) regulate electrical activity between neurons by
controlling the flow of K+ ions down an electrochemical gradient
across neuronal plasma membranes, allowing the membranes to
repolarize after propagating an action potential. Supporting this
theory is the observation of dysfunctional Kv4.2 channels in patients
with epilepsy, a disease marked by increased electrical activity in
the brain. The activity-dependent regulation of Kv4.2 channel
surface expression also may play a role in synaptic integration and
plasticity (Hoffman, 2007). In Alzheimer’s patients, the interaction
of Kv4.2 channels and presenilins has been linked to decreased
neuronal excitability, and, therefore, impaired neurotransmission
and neuronal plasticity (Birnbaum, 2004).
My objective was to make a TfRGFP construct to be used as a
control alongside Kv4.2 in experiments monitoring the expression
of Kv4.2 in the cell membranes of neurons in organotypic slice
cultures.
RESULTS
Kv4.2 ion channel protein
(Kim, 2004)
TfR with bound
transferrin-iron
complex (ebi)
Transferrin receptor (TfR)
ribbon structure (pdb)
Sap I 6993
METHODS
- Used PCR to introduce new restriction
enzyme sites into the sindbis vector.
- Xba1 at n-terminus
- Sph1 at c-terminus
- Ligated TfRGFP insert into the sindbis
vector to create final construct.
- Top10 competent cells were used to
transform the ligation reactions. The
cultures were grown in LB media with
ampicillin at 37°C.
- The Qiagen protocol was followed to
maxi-prep samples.
- The final sample was digested with Xba1
and Sph1; it yielded 3.4 kb fragment and
9.9 kb vector bands.
- A Western blot was performed to check
construct for GFP, using an anti-GFP
antibody.
- Virus preparation using the construct was
conducted utilizing electroporation.
- A titer of the virus concluded the
experiment.
Ahd I 5978
Bsa I 5911
MCS
77 Eco ICRI
77 Sac I
165 Sac II
245 Bst XI
403 Eco RI
409 Sma I
409 Uth SI
409 Xma I
775 Bmg BI
871 Nde I
903 Bsa BI
1002 Afe I
1118 Xcm I
1246 Mfe I
pJPA5TfRGFP.xdna
1807 Pfl MI
1925 Bst Z17I
7233 bp
Unique Sites
nsp2sTfRGFP
Titer:
- TfR 24 hr. conc.: 7.8*10^9 pfu/mL
- TfR 24 hr. dilute: 9.3*10^8 pfu/mL
- TfR 40 hr. dilute: 1.4*10^9 pfu/mL
Zra I 5060
Aat II 5060
Tth 111I 4827
Dra III 4532
Ngo MIV 4429
Nae I 4429
Sfo I 4230
Nar I 4230
Kas I 4230
Bbe I 4230
Avr II 4040
Stu I 4037
Bpl I 3984
Bpl I 3984
Sindbis vector. The blue arrows indicate where the vector
was digested in the multiple cloning site at Sph1 and
Xba1 in preparation for ligation.
Western blot
- anti-GFP
antibody
- The expected
110 kDa band
was identified
2484 Sna BI
2708 Age I
3430 Bsr GI
3442 Not I
3453 Xba I
3459 Sal I
3463 Bsp MI
TfRGFP vector. The blue arrows indicate where restriction
enzyme sites for Xba1 and Sph1 were inserted at the n and c
termini respectively.
Final TfRGFP
construct in
neurons
Further organotypic experiments using
construct nsp2sTfRGFP are now underway
in the Hoffman lab to examine in greater depth
the neurophysiology of Kv4.2 ion channels.
ACKNOWLEDGMENTS
Grant from the NIH Summer
Internship Program in
Biomedical Research
NICHD
Dr. Dax A. Hoffman, Ph.D.
Begum Choudhury, Lin Lin,
Faith Kung, Wei Sun,
Emilie Campanac, Michael Nestor,
and Eun Young Kim