Our work involves characterizing a novel mutation located on the CCR5 receptor gene in human stem cells. cancer es

1. Characterization of the Novel CCR5 Lysine
to Arginine Mutation Using U937 Cells
A. Clarke, A. Lilly, K. Jones, S.
Mares, B. Long, A Calvitti, E.
McCallister

2. The Family IB gave birth to all of her
children naturally, with no
anti-retrovirals.
IIB, the focus of our
research, was exposed
but not infected with
HIV.
Figure 1. A pedigree showing HIV infection in an African American family.

3. HIV Isolate Similarity
HIV isolates from infected family
members were taken and compared to
confirm the mother, IB, as the source of
infection.
Figure 2: Using MacVector, a comparison of genetic distance within the family was modeled.

4. Child IIB CCR5 is a gene that codes for a cell
receptor whose secondary function is
to act as an HIV co-receptor
alongside primary receptor CD4.
Location of
Lys to Arg
(TG5)
mutation.
IIB’s CCR5 gene was
sequenced and the lysine to
arginine mutation was
found.
Figure 3. Amino acid map of the CCR5 receptor showing location of TG5 mutation.

5. Research Questions/Hypothesis
What is the function of the TG5 mutated
gene?
Does the mutation confer HIV resistance?
Hypothesis: The CCR5 mutation TG5 does not
protect a cell from HIV infection.

6. Overview
To characterize the TG5 mutation, we have decided
upon transfection and transduction techniques to
attempt to express the mutation in U937 promonocytic
human stem cell cancer cells.
U937 cells (pictured in slide backgrounds) were chosen because
the cells possess the CCR5 gene but do not express the
extracellular receptor.

7. Transfection
The CCR5 allele containing the Lys to Arg
missense mutation observed in subject IIB
was cloned into pcDNA3.1 (Invitrogen).
The map was confirmed by restriction
endonuclease digestion and analysis.
U937 cells were grown in RPMI with 10%
FBS and 1% Pen-Strep (Invitrogen).
Using Lipofectamine 2000
(Invitrogen), U937 cells were transfected
with pcDNATG5.
Figure 4. Map of constructed transfection plasmid vector pcDNATG5.

8. Transfection A drug study was done to find the
optimal amount of G418 to use in
selection.
Cells were treated with increasing
amounts of G418, from 0μg/μL to
1000μg/μL for a week and counted
three times.
Before counting, cells were treated with
Trypan Blue Stain (Gibco) and dead
cells took in dye.
After cells were transfected they were
treated with 200μg/μL for five weeks.
Ultimately it the transfection was
determined to be unsuccessful.
Figure 5. Graph of G418 drug study U937 cell counts.

9. After transfection proved to be unsuccessful,
Transduction a transduction using pLNCX2 (Clontech)
retroviral vector was started.
First, the vector had to be constructed by
cutting TG5 out of pcDNA with the use of
restriction enzymes XbaI and BamHI.
Oligonucleotide ‘linkers’ were ligated onto
TG5 which converted the BamHI end into a
HindIII site, and the XbaI end into a NotI
site.
This conversion made TG5 compatible for
ligation into the Multiple Cloning Site
(MCS) of pLNCX2.
Figures 6&7. Map of
original pLNCX2
retroviral vector and
MCS sequence.

10. Transduction
Now strands of pLNCX2 containing the
TG5 mutation are being transformed using
competent E.Coli cells to produce sufficient
amounts of plasmid for transduction.
Next, plasmid will be transfected into the
associated packaging cell line PT67 which
will assemble the virus particles.
These particles will be introduced into U937
cultures, and cells will be allowed to grow.
Then successfully transduced cells will be
selected for using the same G418 antibiotics
used in the transfection.
Figure 8. Map of pLNCX2 adapted to show
CCR5 TG5 gene.

11. Future Plans
If the TG5 mutation can be characterized successfully, HIV
infectability may be tested using viral envelope or further testing
if necessary.
We also plan to test any effects of the TG5 mutated gene on both
the CCR5 and CXCR4 (another HIV co-receptor secondary to CD4) receptors.
Provided the mutation is characterized successfully, we will find
another facility to send our cells to in order to test infectability
with actual HIV.
If it is found that the lysine to arginine (TG5) mutation confers
resistance, there are many possibilities for gene therapies, other
treatments, or even a cure.

12. Acknowledgements
Harry Kestler PhD, Rosa Hainaj PhD, John Crooks
PhD, Margaret Gorensek MD, X. Z. Ma MD, Jalpa
Nagisetty, Jessica Jenkins, Anthony George, Katelyn
Witte, Diana Shuman, Kelly Harrison, and Support of
the Lorain County Community College Foundation
Picture Credits
CCR5 map slide - Figure 3:
www.microbytes.com/blog/2010/07/
pLNCX2 map and MCS - Figure 6&7:
http://www.clontech.com/US/Products/Viral_Transduction/Retroviral_Vector_Systems/C
onstitutive_Promoter?sitex=10020:22372:US