ste-A fortiori, even at 10 lm, the four diacylhydrazine ligands did not causesignificant changes in expression of endogenous genes in 293 cells andtherefore should have minimum pleiotropi
Trang 1endogenous gene expression in 293 cells
Siva K Panguluri1, Bing Li2*, Robert E Hormann2and Subba R Palli1
1 Department of Entomology, College of Agriculture, University of Kentucky, Lexington, KY, USA
2 Intrexon Corporation, Norristown, PA, USA
Gene therapy is used to correct a defect in the
expres-sion of a gene by transferring a gene expresexpres-sion
cas-sette containing a promoter, a terminator, and the
coding region of a gene whose absence or defect causes
a disease Current technology uses constitutive
promot-ers, such as the cytomegalovirus promoter, for
expres-sion of transgenes Such an ‘always on’ arrangement is
not desirable because it can exacerbate pleiotropiceffects and also leaves no option for remediation in theevent of medical complications due to transgeneexpression To address this issue, regulated expression
of the additive or corrective gene becomes attractivefor various gene therapy applications Despite extracomplexity, the regulated expression system can
Keywords
diacylhydrazine; ecdysone; gene therapy;
microarray; RSL-1
Correspondence
S R Palli, Department of Entomology,
College of Agriculture, University of
Kentucky, Lexington, KY 40546 USA
Fax: +1 859 323 1120
Tel: +1 859 257 4962
E-mail: rpalli@uky.edu
*Present address
MicroBiotiX, Inc., Worcester, MA, USA
(Received 24 June 2007, revised 13 August
2007, accepted 4 September 2007)
doi:10.1111/j.1742-4658.2007.06089.x
Regulated gene expression may substantially enhance gene therapy lated with structural differences between insect ecdysteroids and mamma-lian steroids, the ecdysteroids appear to have a benign pharmacologywithout adversely interfering with mammalian signaling systems Conse-quently, the ecdysone receptor-based gene switches are attractive for appli-cation in medicine In the present study, the effect of inducers of ecdysonereceptor switches on the expression of endogenous genes in HEK 293 cellswas determined Four ligand chemotypes, represented by a tetrahydroquin-oline (RG-120499), one amidoketone (RG-121150), two ecdysteroids[20-hydroxyecdysone (20E) and ponasterone A (Pon A)], and four diacyl-hydrazines (RG-102240, RG-102277, RG-102398 and RG-100864), weretested in HEK 293 cells The cells were exposed to ligands at concentra-tions of 1 lm (RG-120499) or 10 lm (all others) for 72 h and the totalRNA was isolated and analyzed using microarrays Microarray datashowed that the tetrahydroquinoline ligand, RG-120499 caused cell death
Corre-at concentrCorre-ations‡ 10 lm At 1 lm, this ligand caused changes in theexpression of genes such as TNF, MAF, Rab and Reprimo At 10 lm, theamidoketone, RG-121150, induced changes in the expression of genes such
as v-jun, FBJ and EGR, but was otherwise noninterfering Of the two roids tested, 20E did not affect gene expression, but Pon A caused somechanges in the expression of endogenous genes At lower concentrationspharmacologically relevant for gene therapy, intrinsic gene expressioneffects of ecdysteroids and amidoketones may actually be insignificant
ste-A fortiori, even at 10 lm, the four diacylhydrazine ligands did not causesignificant changes in expression of endogenous genes in 293 cells andtherefore should have minimum pleiotropic effects when used as ligandsfor the ecdysone receptor gene switch
Abbreviations
AMK, amidoketone; qRT-PCR, quantitative real-time reverse transcription PCR; STAT 6, signal transducer and activator of transcription 6; THQ, tetrahydroquinoline.
Trang 2substantially raise the level of safety and might even
be essential to control the expression levels of proteins
that have narrow therapeutic indices, such as
cyto-kinins and hormones
The main purpose of a regulated gene expression
system is to control the timing and levels of transgene
expression in vivo Whether the expressed protein
remains within the cell or, more commonly, is secreted
and⁄ or distributed in extracellular compartments, it
will undergo elimination according to pharmacokinetc
principles [1] Thus, too little protein will be
subthera-peutic, too much will be potentially toxic Therefore, a
successful therapy would be characterized by regulated
expression of the transgene finely tuned to the
chang-ing clinical state of a patient
Several gene switches have been developed for
regu-lating the expression of transgenes in humans [2]
More specifically, ecdysone receptor (EcR) gene
switches for medicinal purposes have been reported by
several laboratories [3–9] Among these switches,
EcR-based gene switches display particularly low basal
activity in the absence of an inducer and strong
induc-ible activity in the presence of an inducer [2,8,10]
The relative structural dissimilarity of ecdysteroidsand mammalian steroids might suggest that binding ofthe former to vertebrate steroid receptors would be tooweak for pharmacological effects, particularly adverseones In support of this proposition, humans consumesignificant amounts of phytoecdysteroids contained indietary vegetables seemingly without any apparentdetrimental effects [11] However, Oehme et al [12]reported recently that ecdysteroids and ecdysonemimics can induce and⁄ or suppress endogenous genes
in RKO and other mammalian cells and promoteapoptosis
A more comprehensive understanding of possiblepleiotropic effects of ligand inducers and⁄ or the switchcomponents is essential for successful use of the EcRgene switch for in vivo applications such as gene ther-apy The diacylhydrazine [13,14] nonsteroidal ecdysoneagonists, such as Rheoswitch ligand 1 (RSL-1; Fig 1),are reported to be an excellent inducer for EcR geneswitches, supporting up to 9000-fold induction ofreporter activity [15] Other steroidal ligands, such asponasterone A (Pon A; Fig 1), have also beenreported as potential inducers of EcR-based gene
O
O O
Diacylhydrazines (DAH) RG-100864 R 1 =Cl R 2 =H
R = OH
R = H
RG-102398 R 1 =2-CH 3 , 3-OCH 3
R 2 =3,5-di-CH 3 RG-102240 R 1 =2-CH 2 CH 3 , 3-OCH 3
R 2 =3,5-di-CH 3 RG-102277 R 1 =2-CH 2 CH 3 , 3-OCH 3
R 2 =3-CH 3 , 5-C(O)NH 2
N O
HN F
F
F
N H
O O
Fig 1 EcR ligands analyzed by microarray and qRT-PCR analysis.
Trang 3switches [8,14] In addition, other chemotypes, such
as amidoketones (AMK) and tetrahydroquinolines
(THQ), are also being developed as inducers of EcR
gene switches [16–20] (Fig 1)
The main goal of the present study was to determine
the intrinsic gene expression effects of EcR switch
in-ducers in mammalian cells We studied the effect of
eight EcR ligands: four diacylhydrazines, two
ecdyster-oids, one THQ and one AMK ligand (Fig 1) on the
expression of endogenous genes in HEK 293 cells
using microarray and quantitative real-time reverse
transcription PCR (qRT-PCR) THQ ligand caused
changes in the expression of genes such as TNF, MAF,
Rab and Reprimo The AMK ligand induced changes
in the expression of genes such as v-jun, FBJ and
EGR 20-Hydroxyecdysone (20E) did not affect gene
expression, but Pon A caused some changes in the
expression of endogenous genes At lower ligand
con-centrations applicable for therapeutic use, potential
pleiotropic effects may or may not be observed The
four diacylhydrazine ligands did not cause significant
changes in the expression of endogenous genes
Results and Discussion
THQ ligand, RG-120499, affects 293 cells via many
pathways
Incubation of 293 cells with the THQ compound
RG-120499 at 10 lm for 3 days resulted in the death of
70% of the cells as indicated by observation of cell
morphology To determine the possible genes and
path-ways that are affected by this ligand, we performed a
microarray experiment using RNA isolated from
293 cells treated with 1 lm RG-120499 A total of 1171
genes were up-regulated and 443 genes were
down-regulated in 293 cells treated with this compound
compared to the cells treated with dimethylsulfoxide
(Fig 2A) Among these, 115 genes showed P£ 0.01
with a two-fold or more greater in expression levels in
ligand-treated cells compared to the levels in
dimethyl-sulfoxide-treated cells (Table 1) Among these 115
genes, 55 genes showed signal detection values of more
than 100 We selected v-maf, TNF, PNAS13, Rab,
Rep-rimo, DNAH and KIF9 genes for PCR The
qRT-PCR data (Fig 2B,C) showed that v-maf, TNF,
PNAS13and DNAH mRNA levels increased, and Rab
and Reprimo mRNA levels decreased in RG-120499
treated cells, compared to the levels in
dimethylsulfox-ide-treated cells The microarray and qRT-PCR data
showed perfect correlation for these six genes The
two-fold down-regulation of the KIF9 gene observed in
microarray analyses was not confirmed by qRT-PCR
because this method showed a ten-fold up-regulation ofthis gene in the presence of RG-102499
MAF [v-maf musculoaponeurotic fibrosarcomaoncogene homolog (avian)] is a basic-leucine zippertranscription factor that plays crucial roles in gene reg-ulation, differentiation, oncogenesis and development
in many organisms [21] v-maf is a viral oncogeneencoding a leucine zipper motif that forms heterodi-mers with the protein products of maf-related genes orother proteins such as fos, jun and myc oncogenes thathave leucine zipper motifs [22] Our microarray andqRT-PCR data showed that, in 293 cells, RG-120499up-regulates v-maf gene expression by 2.4- and2.3-fold, respectively Nishizawa et al [22] have dem-onstrated that the human cellular counterpart of thev-maf (c-maf) gene is conserved across species Addi-tionally, Massrieh et al [21] reported that the MAFtranscription factor transcript levels are induced byproinflammatory cytokines in PHM1-31 myometrialcells and that MAF transcription factor mRNA israpidly induced by IL-1B and TNF in primarymyometrial and PHM1-13 cells Our data also indicateup-regulation of the TNF gene in microarray andqRT-PCR data (by 2.2- and 5.5-fold, respectively), anobservation consistent with TNF-induced up-regulation
of MAF transcript levels Zheng et al [23] reportedthat the tumor necrosis factor (TNF) can mediatemature T-cell receptor-induced apoptosis through thep75 TNF receptor This may be the possible reasonfor the death of 293 cells when treated with 10 lmRG-120499 ligand
In addition to the aforementioned up-regulatedgenes, two genes, namely, Rab and Reprimo, weredown-regulated by this THQ ligand in 293 cells, asobserved by both microarray and qRT-PCR tech-niques The Rab proteins constitute a subfamily ofRas-related GTP-binding proteins that are localized indistinct intracellular compartments [24] Mutations inthe Rab gene can alter the morphology of entire organ-elles by blocking protein transport along the exocyticand endocytic pathways because Rab proteins plays akey role in membrane trafficking [25] Barbosa et al.[26] reported that mutations in the Rab gene(s) cancause irregularities in the protein transport machineryleading to the formation of giant lysosomes in mousebeige (bg) mutant and other mutant mice It has beensuggested that Chediak–Higashi syndrome, a rareautosomal recessive disorder in humans, is the conse-quence of a mutation to a homolog of bg Our micro-array and qRT-PCR data indicate that the rab gene wasdown-regulated in 293 cells by two- and 23-fold, respec-tively, by the THQ, RG-120499 Mutation studies of theRabgene suggest that the Rab gene down-regulation by
Trang 4this ligand may be a reason for cell death in addition to
modulation of the v-maf and TNF genes
Tumor suppressor genes that encode transcriptional
factors can affect a variety of cellular mechanisms
underlying growth, differentiation, and apoptosis
[27,28] Also, when cells were exposed to DNA
dam-age-inducing agents or other noxious stress, the p53
protein, which is the most commonly mutated gene in
human cancer, is induced and⁄ or activated, resulting in
cell cycle arrest or apoptosis [29–31] Reprimo is a
highly glycosylated protein, which will localize in the
cytoplasm and induce G2 arrest of the cell cycle when
expressed ectopically [32] In the present study, it was
observed that RG-120499 down-regulates the Reprimo
gene by two-fold as measured by both microarray and
qRT-PCR techniques From these observations and
previous reports, we suggest that the down-regulation
of the Reprimo gene may cause loss of DNA repair,which, in turn, is passed on to the next generations,thereby accumulating DNA defects RG-120499 modu-lates the expression of other genes as well ThePNAS123 gene (transformation related protein 11) isup-regulated by 2.7- and 4.3-fold as measured bymicroarray and qRT-PCR, respectively The pathways
in which these genes are involved are not known
RG-120499 also triggers five- and 23-fold up-regulation ofthe DNAH (human axonemal dynein heavy chain)gene as measured by microarray and qRT-PCR tech-niques, respectively DNAH is a microtubule associ-ated motor protein that moves cilia and flagella[33,34] Afzelius [35,36] showed that patients sufferingfrom Kartagener syndrome have cilia lacking dynein
Trang 5arms This disease is characterized by chronic
respira-tory tract infections, altered position of internal
organs, and infertility arising from immotile sperm
Milisav and Affara [37] reported that the human
dynein-related gene DNEL2 may play an important
role specifically in sperm motility and is not involved
in the movement of cilia In summary, the use of the
THQ gene switch ligand RG-120499 may cause
signifi-cant changes in the expression of host genes
AMK, RG-121150, affects gene expression in
293 cells
The affect of the AMK RG-121150 on gene expression
in 293 cells was analyzed using microarray and
qRT-PCR In microarray analysis, a total of 636 genes were
up-regulated and 604 genes were down-regulated by
this ligand (Fig 3A) Among these genes, 71 genes
showed P£ 0.01 and a two-fold or greater change in
expression (Table 1) Among 71 genes, 24 genes
showed signal detection values greater than 100
Among these are hypothetical proteins, nuclear
pro-teins, transcriptional factors, glycogen phosphorylase,
hormone degrading enzymes, kinases and some solute
carrier proteins To validate microarray data with
qRT-PCR, the primers for early growth response gene
(EGR), FBJ, v-jun, and a hypothetical protein genewere designed For three of the four genes, the micro-array data was confirmed by qRT-PCR The datashowed that the hypothetical protein gene was up-regu-lated by two- and 1.5-fold in microarray and qRT-PCRexperiments, respectively EGR and v-jun showed sup-pression of their expression by the ligand in analyses byboth methods (Fig 3B,C) The gene FBJ mRNA levelswere down-regulated in microarray experiments and up-regulated in qRT-PCR experiments
The EGR gene product is a transcription factor thatplays a role in differentiation and growth EGR genesare transiently and coordinately induced upon activa-tion of peripheral blood T lymphocytes [38,39] TheseEGR genes are also expressed in a wide range of celltypes, including lymphoid cells, myeloid cells such asthymocytes, B cells, monocytes, and nonlymphoid cellssuch as fibroblasts, kidney cells and neurons [40,41].Huang et al [42,43] showed that the expression of theEGR gene exogenously in various tumor cells unex-pectedly and markedly reduces growth and tumorige-nicity, whereas the suppression of endogenous EGR
by antisense RNA enhances growth and promotesphenotypic transformation From our microarray andqRT-PCR data, the AMK RG-121150 caused down-regulation of this EGR gene by five- and six-fold,
Table 1 Differentially expressed genes in 293 cells treated with different ligands.
Up-regulated Down-regulated Total Up-regulated Down-regulated Total
Trang 6respectively, indicating a possible effect RG-121150 on
this pathway Overall, these studies showed that AMK
ligand RG-121150 could cause significant changes in
the expression of genes in host cells However, the
cor-responding toxicological potential could be
substan-tially mitigated or even erased by the likelihood that
actual gene therapy dosage would be significantly
lower than the 10 lm studied here Likewise, judicious
choice of the specific member of the AMK chemotype
may confer intrinsic benignity
Ecdysteroid ligand, 20E, shows little effect on
293 cells but Pon A affects cell division
Two steroid ligands Pon A and 20E were tested in
293 cells to determine their effect on gene expression
using microarray and qRT-PCR In 20E-treated
293 cells, a total of 542 genes were up-regulated and
627 genes were down-regulated compared to sulfoxide-treated cells (Fig 4A) Out of these, 51 genesshowed P£ 0.01 and a change in gene expression oftwo-fold or greater (Table 1) Among these 51 genes,only five genes had signal detection values greater than
dimethyl-100 We selected signal transducer and activator oftranscription 6 (STAT6) genes to perform qRT-PCRanalyses Although the STAT6 gene was down-regu-lated by 20E in microarray analysis, the qRT-PCRanalyses showed that the mRNA levels did not change
in treated cells compared to untreated cells(Fig 4B,C) Experiments in our laboratory (S R Palli,unpublished results) showed that, in mammalian cells,20E does not induce reporter genes placed under the
k Amido ketone Amido ketone Amido ketone
Fig 3 AMK RG-121150 affects endogene expression in 293 cells (A) The V-plot of differentially expressed genes from microarray analysis The P-values of t-test are plotted against fold suppression or induction The horizontal bar in the plot represents the nominal significant level 0.001 for the t-test under the assumption that each gene has a unique variance The vertical bars represent the genes that are minimum of two- fold up- or down-regulated compared to the control dimethylsulfoxide (DMSO) (B) The signal values of hypothetical protein, EGR, v-jun and FBJ genes from microarray The signal values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide (C) The relative expressions of hypothetical protein, EGR, v-jun and FBJ gene transcript levels in qRT-PCR The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide.
Trang 7control of the EcR gene switch The microarray data
corroborate evidence for an overall benign influence of
20E on gene expression and metabolism in mammalian
cells [44] It is conceivable that 20E is excluded by
mammalian cells, or perhaps some other factors in the
mammalian cells inhibit the activity of 20E
A total of 639 genes were up-regulated and 638 were
down-regulated in 293 cells treated with Pon A
com-pared to the cells treated with dimethylsulfoxide
(Fig 5A) Among these, 41 genes showed P£ 0.01 and
two-fold or greater induction (up⁄ down-regulated;
Table 1) Only three genes showed signal detection
val-ues greater than 100 Out of these three genes, we
selected tousled-like kinase (Tlk) gene for qRT-PCR
analysis The data from both methods showed that the
Tlk gene was induced by Pon A by two-fold
(Fig 5B,C) In proliferating human cells, Tlks are
maximally active during the S phase but rapidly vated in response to inhibitors of DNA replication[45] Sillje and Nigg [46] showed that the Asf1 (anti-silencing function 1) proteins are phosphorylated byTlks both in vivo and in vitro during the S phase anddephosphorylated with inactivation of Tlks Constan-tino et al [47] used the ecdysone inducible gene expres-sion system in hematopoietic cells and found that thetwo steroids, muristerone A and Pon A, altered thesignaling pathways induced by IL-3 in the pro-B cell-line, Ba⁄ F3 Indeed, they also showed that these ecdy-steroids potentiate the IL-3-dependent activation ofthe PI 3-kinase⁄ Akt pathway, an effect that could ulti-mately interfere with the growth, and⁄ or survival ofthese cells Our data do not reveal any affect of Pon A
inacti-on the PI 3-kinase⁄ Akt pathway in 293 cells A ble reason is that these genes might be expressed only
e Expression 0.002
0.001
0 -2.86
20E DMSO
STAT6
20E DMSO
Trang 8analy-in a particular type of cells, such as hematopoietic cells,
but not 293 cells These observations, in conjunction
with our microarray and qRT-PCR data, indicate that
Pon A may affect cell division in mammalian cells
Diacylhydrazine ligands do not induce significant
changes in gene expression
Diacylhydrazine nonsteroidal ligands have been
sub-jected to extensive studies concerning their
toxicologi-cal effects on vertebrates for the purposes of
Environmental Protection Agency registration as
com-mercial insecticides [48,49] We selected four
represen-tatives from the diacylhydrazine gene switch
chemotype for evaluation of their effects on expression
of endogenous genes in 293 cells: the lepidopteran
insecticide, methoxyfenozide (RG-102398); the
coleopt-eran insecticide, halofenozide (RG-100864); the
Rheo-Switch ligand RSL-1 (RG-102240, also known as
GSTM-E); and, finally, a more polar and water-solublevariant of RSL-1, namely RG-102277 First, we deter-mined the effect of dimethylsulfoxide itself on theexpression of genes in 293 cells The expressions of atotal of 43 genes were modulated with P£ 0.01 with atwo-fold change in expression Among the affectedgenes, 38 were up-regulated and five of them weredown-regulated Interestingly, only 13 up-regulatedgenes and one down-regulated gene showed signaldetection values of more than 100 in the dimethylsulf-oxide-treated 293 cells In our experience, the genesthat show signal detection values less than 100 are notreliable indicators of gene expression; therefore, we didnot consider these in our analyses
Cells treated with the diacylhydrazine RG-102240resulted in up-regulation of a total of 865 genes; on theother hand, 411 genes were down-regulated in 293 cellstreated with RG-102240 (Fig 6A) From these, we con-sidered for further analysis only the genes with P£ 0.01
e Expression 0.000502
PonADMSO
TLK
PonADMSO
1 x1.5 x4 x8 x16
Fig 5 Steroidal ligand Pon A may affect cell division in 293 cells (A) The V-plot of differentially expressed genes from microarray analysis The P-values of t-test are plotted against fold suppression or induction The horizontal bar in the plot represents the nominal significant level 0.001 for the t-test under the assumption that each gene has a unique variance The vertical bars represent the genes that are minimum of two-fold up- or down-regulated compared to the control dimethylsulfoxide (DMSO) (B) The signal values of Tlk gene from microarray The signal values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar rep- resents the fold changes with this ligand against dimethylsulfoxide (C) The relative expression of Tlk transcripts as determined by qRT-PCR The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide.
Trang 9with a two-fold change in expression compared to the
expression in dimethylsulfoxide-treated cells A total of
46 genes met these criteria However, only six of these
genes showed signal detection values greater than 100
(Table 1) The annotations for these genes were
devel-oped by using NIH david ease software (http://david
niaid.nih.gov/david/ease.htm) Only two out of six were
annotated; these were identified as DAB2 interacting
protein gene and kinesin family member-9 (KIF9)
The reliability of microarray results depends on
sev-eral factors such as array production, RNA extraction,
probe labeling, hybridization conditions and image
analysis [50–53] Therefore, the genes identified as
dif-ferentially expressed by this method must be validated
with another method such as qRT-PCR, which is
quantitative, rapid, and requires 1000-fold less RNA
than conventional assays [54] For this reason, wedesigned primers for DAB2 interacting protein geneand KIF9 from their cDNA sequences for qRT-PCRanalysis Their expression levels were measured indimethylsulfoxide- and RG-102240-treated 293 cellsand compared to microarray data The DAB2 interact-ing protein gene showed two-fold down-regulation bymicroarray and a four-fold increase by qRT-PCRanalysis in RG-102240-treated cells compared tothe expression in dimethylsulfoxide-treated cells(Fig 6B,C) The KIF9 gene showed 3.9-fold down-reg-ulation by microarray and 2.8-fold down-regulation byqRT-PCR analysis in RG-102240-treated cells com-pared to the expression in dimethylsulfoxide-treatedcells (Fig 6B,C) The Drosophila melanogaster Dab(Disabled) interacting protein is the mammalian ortho-
0.0040.0030.0020.0010
Trang 10logue of D melanogaster DabIP The DabIP
partici-pates in a signaling complex containing various
pro-teins involved in brain development as well as other
aspects of adult brain function [55] Although the
microarray showed two-fold down-regulation of the
Dab gene, the qRT-PCR data showed four-fold
up-regulation The reasons for difference in the fold
regulation between the two techniques are not readily
apparent KIF9 protein is found to interact with
Ras-like GTPase Gem and is involved in cell shape
remod-eling [56] Both microarray data and qRT-PCR data,
showed similar results (3.9-fold and 2.8-fold
down-reg-ulation, respectively) in KIF9 gene expression levels
Over-expression of the KIF9 mutant did not cause any
significant difference in Gem-induced cell elongation
and it does not appear to be essential for the
Gem-induced phenotypic changes [56] From these tions, it can be concluded that, although the RSL-1ligand down-regulates the KIF9 gene, there may not besignificant changes in cell shape remodeling
observa-Treatment of HEK 293 cells with RG-102398resulted in a total of 598 down-regulated and 388up-regulated genes (Fig 7A) Among these, 52 genesindicated P£ 0.01 and a change in expression of two-fold or greater compared to dimethylsulfoxide-treatedcells (Table 1) Only three genes [ribosomal protein L13(RPL), hypothetical protein gene FLJ38705 and leptinreceptor] out of these 52 showed signal detection valuesgreater than 100 The qRT-PCR and microarray anal-ysis of RPL gene expression are weakly correlated.The RPL gene was down-regulated by two-fold bymicroarray, but was up-regulated 1.25-fold by
-2.27-2.05
Zink Finger RPL
Zink Finger RPL