Protocols and Applications Guide (US Letter Size) - Promega

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conclude that methylation plays a dual role in regulating
hTERT expression. CTCF will bind to the first exon of
hTERT when the hTERT CpG island is not methylated,
resulting in downregulation of hTERT expression.
PubMed Number: 17267411
Wu, S. et al. (2006) Reversal of the malignant phenotype of
cervical cancer CaSki cells through adeno-associated
virus-mediated delivery of HPV16 E7 antisense RNA.
Cancer Res. 12, 2032–7.
The coding sequence of the Human Papilloma Virus
(HPV16) E7 oncogene was isolated after purification of
total RNA from CaSki cells, RT-PCR, subsequent PCR and
cloning into the pGEM®-T Easy Vector. To test the
effectiveness of antisense HPV16 E7 therapy against cervical
cancer, an adeno-associated virus vector was constructed
using this coding sequence and used to transfer the
antisense construct of the E7 coding sequence into CaSki
cervical cancer cells.
PubMed Number: 16609012
pTARGET Mammalian Expression Vector System
The pTARGET Mammalian Expression Vector System
(Cat.# A1410) is a convenient system to clone PCR products
and express cloned PCR products in mammalian cells. As
with the pGEM®-T and pGEM®-T Easy Vector Systems,
the pTARGET Vector is supplied already linearized with
single T overhangs (Figure 13.3). These single 3´ T
overhangs at the insertion site greatly improve the
efficiency of ligation of a PCR product into the plasmid.
The pTARGET Vector also contains a modified version of
the coding sequence of the α peptide of β-galactosidase,
which allows recombinants to be selected using blue/white
screening.
Figure 13.3. pTARGET Vector circle map.
The pTARGET Vector carries the human cytomegalovirus
(CMV) immediate-early enhancer/promoter region to
promote constitutive expression of cloned DNA inserts in
mammalian cells. This vector also contains the neomycin
phosphotransferase gene, a selectable marker for
mammalian cells. The pTARGET Vector can be used for
transient expression or for stable expression by selecting
transfected cells with the antibiotic G-418. Like the
Protocols & Applications Guide
www.promega.com
rev. 3/09
pGEM®-T or pGEM®-T Easy Vectors, inserts of several
kilobases can be cloned in and expressed from the
pTARGET Vector (Sakakida et al. 2005;
Le Gall et al. 2003).
Additional Resources for the pTARGET Mammalian
Expression Vector System
Technical Bulletins and Manuals
TM044 pTARGET Mammalian Expression Vector
System Technical Manual
(www.promega.com
/tbs/tm044/tm044.html)
Promega Publications
PN082 Technically Speaking: T-vector cloning
(www.promega.com
/pnotes/82/10203_24/10203_24.html)
PN058 pTARGET Vector: A new mammalian
expression T-vector
(www.promega.com
/pnotes/58/5189a/5189a.html)
Online Tools
pTARGET Mammalian Expression Vector sequence
(www.promega.com/vectors/ptarget.txt)
Citations
Treeck, O. et al. (2007) Novel estrogen receptor beta
transcript variants identified in human breast cancer cells
affect cell growth and apoptosis of COS-1 cells. Mol. Cell.
Endocrinol. 264, 50–60.
This study identified two novel transcript variants of the
estrogen receptor ERβ that were expressed in the
ERα-negative breast cancer cell line MDA-MD-231. These
variants were identified after amplification of ERβ
transcripts from the breast cancer cell line by RT-PCR. The
amplification products were then excised from gels and
subcloned into the pTARGET Mammalian Expression
Vector prior to sequencing. COS1 cells, which do not
express the estrogen receptor, were then stably transfected
with full-length ERβ or one of the splice variants, and the
effects on cell proliferation, apoptosis and estrogen response
were evaluated. In COS1 cells expressing either ERβ or the
transcript variants, cell proliferation decreased and basal
apoptosis (caspase-3/7 activity) increased, compared to
cells transfected with vector alone. Exposure to therapeutic
doses of tamoxifen induced apoptosis in cells expressing
the full-length ERβ but not in cells expressing either of the
variant isoforms.
PubMed Number: 17095148
Guyonnet-Duperat, V. et al. (2006) Functional implication
of an ARG307GLY substitution in corticosteroid binding
globulin, a candidate gene for a QTL associated with
cortisol variability and obesity. Genetics 173, 2143–9.
In this study, the effects of amino acid substitutions in
porcine corticosteroid-binding globulin gene (Cbg) were
tested on CBG binding and affinity. Cbg cDNA was
obtained by reverse transcribing pig liver total RNA using
M-MLV Reverse Transcriptase followed by PCR. The
PROTOCOLS & APPLICATIONS GUIDE 13-5