Open Access

Effect of exogenous transcription factors integration sites on safety and pluripotency of induced pluripotent stem cells


Cite

Figure 1

Schematic diagram of nested iPCR.
Schematic diagram of nested iPCR.

Figure 2

Nested iPCR. (A) Restriction digestion of genomic DNA. M: 1 kb marker. (B) The result of nested iPCR. M: 1 kb marker; Oct4, Sox2, Klf4, c-Myc: exogenous TFs.
Nested iPCR. (A) Restriction digestion of genomic DNA. M: 1 kb marker. (B) The result of nested iPCR. M: 1 kb marker; Oct4, Sox2, Klf4, c-Myc: exogenous TFs.

Figure 3

Expression profiles of flanking genes from microarray analysis. (A) Expression profiles of Oct4 flanking genes. (B) Expression profiles of Sox2 flanking genes. (C) Expression profiles of Klf4 flanking genes. (D) Expression profiles of c-Myc flanking genes.
Expression profiles of flanking genes from microarray analysis. (A) Expression profiles of Oct4 flanking genes. (B) Expression profiles of Sox2 flanking genes. (C) Expression profiles of Klf4 flanking genes. (D) Expression profiles of c-Myc flanking genes.

Figure 4

The GO classification of flanking genes.
The GO classification of flanking genes.

The molecular functions of flanking genes.

GenesFunctionMainly Process Involved
Phlda1protein bindingcell apoptosis; programmed cell death
Krr1RNA binding; POLY-A bindingbiosynthesis of ribosomes; rRNA process
Vat1Zn2+ binding; oxioreductase activitynegative regulation of mitochondrial fusion; redox
Lsm10RNA binding; histone precursor mRNA bindingpositive regulation of mitosis from G1 to S phase
Stk40kinase activation; ATP bindingembryonic ectoderm differentiation; glucose metabolism
Nanos3RNA binding; Zn2+ bindingoogenesis; spermatogenesis
Zswim4metal ion binding; Zn2+ bindingbiochemical process
Samd4POLY-A bindingnegative regulation process of synapse; translation
Alox5approtein binding; enzyme bindingmetabolism process of leukotrienes
Medagcatalysis; bindingadipocyte differentiation
Rassf10catalysis; bindingbiochemical processes; signal transduction
ArntlDNA binding; RNA binding; protein bindingregulation of gene expression related to circadian clock
Hspa4ATP binding; protein complex bindingintroduction of mitrochondrial outer membrance proteins
Zcchc10metal ion bindingbiochemical processes
Ankrd7catalysis; bindingsupport cell maturation
Kcnd2ion channel activationK+ transmembrane transport
Dctn6kinetic protein bindingmotochondrial cavity
Olfr456; Olfr455protein bindingolfactory receptors; signal transduction
Atp13a3ATP binding; ATP enzyme activationNa+ transmembrane transport; cation transport
Tmem44transmembrane protein bindingbiochemical processes
Kbtbd11N/Atranscriptional regulation
Myom2components of cytoskeleton structuralmuscle contraction
Jagn1molecular function (basic cell activity)neutrophil-fungal-induced mediated immune immune response response;
Frylprotein bindingbiochemical processes
Cops7btransmembrane protein functional moleculedeubiquitination
Nppcpeptide hormone receptor binding; hormone activityvasodilation; endochondral growth
Aldh8alaldehyde dehydrogenase activityretinoic acid metabolism
Sgk1ATP binding; protein binding; kinase activitypositive negative regulation regulation of of Naapoptosis + transportation;
Abcd3ATP binding; protein bindingtransmembrane transportation; long chain fatty acid metabolism
Arhgap29activity of GDP enzyme; protein bindingintercellular signal transduction
Cdh6Ca2+ binding; metal ion bindingcell adhesion
Slcl3a3cotransport activity; transmembrane transport activitysuccinic acid transportation; Na+ transportation
Rarbbinding; retinoic acid receptorpositive digestive regulation system development; of cell proliferation; neurogenesis
Thrbthyroxine; activity of receptor thyroxineintracellular receptor of signaling pathway; organ morphonenesis
Mettl15activity of methyltransferasemethylation; ribosomal methylation
Kcna4K+ binding; ion channel activationion transport; K+ transmembrane transport

The identified integration sites and their flanking genes.

Flanking Genes and Their Distance
Reprogramming FactorsiPSCsChromosome No. of Integration SiteGenBank Acc. No.eIntegration SitGene5Distance (kb)3Distance (kb)
Sox2IP14D-110NC_000076.6intergenicPhlda18.3Krr1450.0
10NC_000076.6intergenicAldh8a1301.0Sgk1185.0
IP14D-61NC_000076.6intergenicCops7b28.2Nppc35.7
IP14D-10111NC_000077.6intronsVat1
8NC_000074.6intergenicNanos320.9Zswim414.3
IP14D-115NC_000081.6intergenicCdh62390.0LOC101055956461.1
14NC_000080.6intergenicRarb287.3Thrb554.7
Oct4IP14D-616NC_000082.6intergenicAtp13a3111.0Tmem4414.1
7NC_000073.6intronsSamd4
IP14D-1015NC_000071.6intergenicAlox5ap102.8Medag15.7
2NC_000068.6intronsSlc13a3
7NC_000073.6intergenicRassf10196.2Amtl128.1
IP14D-18NC_000074.6intergenicKbtbd112.7Myom225.4
5NC_000071.6intergenicFryl57.7LOC1052441505.8
Klf46NC_000072.6intronsJagn1
IP14D-611NC_000077.6intergenicHspa46.3Zcche1018.1
IP14D-1014NC_000070.6intergenicLsm1013.8Stk406.2
6NC_000072.6intergenicAnkrd71579.0Kcnd2766.4
IP14D-13NC_000069.6intergenicAbcd3126.2Arhgap2933.7
c-Myc6NC_000072.6intergenicOlfr4561.4Olfr45549.3
IP14D-68NC_000074.6intronsDctn6
IP14D-1016NC_000072.6intergenicKcna4459.7Mettl151289.0

The primer sequences of nested inverse polymerase chain reaction

PrimerSequences (5’>3’)
First iPCR-FAAA ATA ATA ATA ACC GGG CAG GCC A
First iPCR-Sox2-RCCT TCT TCA TGA GCG TCT TGG TTT T
First iPCR-Oct4-RGTG TCC CTG TAG CCT CAT ACT CTT C
First iPCR-Klf4-RCTT TGC TAA CAC TGA TGA CCG AAG G
First iPCR-c-Myc-RTCT TCT CCA CAG ACA CCA CAT CAA T
Second iPCR-FCAG CAC AGT GGT CGA CGA TAA AAT A
Second iPCR-Sox2-RTTC AGC TCC GTC TCC ATC ATG TTA T
Second iPCR-Oct4-RTTT GCA TAT CTC CTG AAG GTT CTC A
Second iPCR-Klf4-RGGG TTA GCG AGT TGG AAA GGA TAA A
Second iPCR-c-Myc-RCCTCCAAGTAACTCGGTCATCATCT
eISSN:
1311-0160
Language:
English
Publication timeframe:
2 times per year
Journal Subjects:
Medicine, Basic Medical Science, other