, SLC26A4 HEK 293. The construction of wild-type SLC26A4 gene expression vector and the expression in HEK293 cells

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1 SLC26A4 HEK (100853) 2 ( ) SLC26A4 (pegfp enhanced green fluorescent protein) SLC26A4 pegfp-slc26a4 HEK293 pegfp-slc26a4 HEK293 - (RT-PCR) SLC26A4 mrna Western Blot ( ) Pendrin SLC26A4 GenBank SLC26A4 pegfp-slc26a4 HEK RT-PCR SLC26A4 Western Blot 87 kda SLC26A4 pegfp HEK293 SLC26A4; ; R349.6 R394.8 A (2011) The construction of wild-type SLC26A4 gene expression vector and the expression in HEK293 cells WU Jian 1 ZHANG Guo-zheng 2 YANG Shi-ming 1 DAI Pu 1 YUAN Yong-Yi 1 1 Department of Otolaryngology Head and Neck Surgery Institute of Otolaryngology Chinese PLA Genernal Hospital Beijing China 2 Department of Otolaryngology Third Hospital of Hebei Medical University Shijiazhuang China Corresponding author: YUAN Yong-yi linc301@yahoo.com Abstract Objective To construct a eukaryotic expression vector containing the wild-type SLC26A4 gene and green fluorescent protein gene for clinical deafness diagnosis and prenatal diagnosis about SLC26A4 -related deafness. Methods Using the Gene recombination and restriction enzyme digestion and gene sequencing to construct and identify the eukaryotic expression plasmid of pegfp-slc26a4. Using fluorescence microscope to observe the expression of transfected HEK293 cells by liposome using reverse transcription-polymerase chain reaction (RT-PCR) to detect the mrna and using Western Blot to detect SLC26A4 protein expression. Results The positive recombinant gene contain the SLC26A4 fragment and the sequence were the same sequence as on the GenBank. After being transfected Into HEK293 cells The green fluorescent can be observed and The SLC26A4 bands can be amplified. The 87kDa-sized protein was detected by Western Blot. Conclusion we successfully constructed the eukaryotic expression vector containing wild-type SLC26A4 and EGFP gene and the vector can express in mammalian HEK293 cell lines. Key Words SLC26A4; Plasmid; Expression : (2009B34) : 2008 ( ); % : : linc301@yahoo.com

2 78 Chinese Journal of Otology Vol. 9 No μl PCR PCR [1] Yuan [2] bp 54.93% 1.2 PCR SLC26A % GJB2 : 10 5 μl 100 BSA Pendred 1.3 PCR SLC26A ng PCR 100 ng T4 DNA 2 Pendrin Pendrin SO - 4 Ligase (T4 DNA ) 4 HCO 3- I - Cl Pendrin DH5α SLC26A4 200 μl HEK293 (human enbryonic kidney cell line ) 30 min kda SLC26A μl RNA 20 mmol / L MgSO 4 AMP ( ) cdna PCR : LB Xho -F:5' -CCGCTCGAGATGGCAGCGCCAG GCGGCAG-3' Kpn -R:5' -GGGGTACCGTGGATGCAAGTGT h DNA pegfp (enhanced green fluorescent protein)-slc26a4 ACGCATAG-3' NCBI GenBank SLC26A4 cdna Primer Premier Kpn( 1 / 10) 20 μl h PCR ( ) 1 μg Invitrogen Invtrogen PCR : 10 Pfx50 PCR Mix ( MgCl 2 ) 5 μl pegfp-slc26a4 HEK293 mmol / L dntp ( ) Mix 1.5 μl Primer Mix 1.5 μl DNA 3 μl Pfx50 DNA μl 50 μl 6 : s s s 72 3 s s Xho Kpn (bovin serum alburmin ) 0.5 μl SLC26A4 pegfp-n1 PCR 2 μl Xho 1 μl Pendred Kpn 1 μl 20 μl 37 1 h 4% ~ 10% Pendred DNA ( pegfp-n1 ) 2 μl 90 s l ~ 2 min 800 μl LB min 0.5 ~ 1 μg Xho / ml HEK293 80% DNA Lipofectamine TM 2000

3 (Invitrogen) 1 μg 3μl 2 SDS ( ) DMEM (Dulbecco's Modified Eagle Medium) min ( ) 100 μl 20 min SDS-PAGE (SDS- ) DNA- Lipofectamine TM 2000 PVDF ( ) μl 6 h Pendrin h μl 24 h 4% TBS-Tween (Tween 20 Tris ) 40 min 10 min 3 ( ) IgG min TBS-Tween RT-PCR ( - ) Western 10 min 3 Blot ( ) pegfp-slc26a4 24 h 0.25% 800 r / min IgG ( ) 1.5 ml Eppendorf RT-PCR Western Blot RT-PCR SLC26A4 / β-actin Trizol (Invitrogen) RNA 3 cdna 2 RT-PCR (Invitrogen) PCR (500 ~ 750 bp) mrna PCR : 95 5 min s s s 72 5 min 40 : 5'-GAATTCCACCATGGCAGCCG- GGAGCATCA-3' : 5'-TCTAGATCAGCTCTTAGCAGA- CATTGG-3' Western Blot X β-actin (β- ) Labworks Xho Kpn 2360 bp pegfp -SLC26A4 NCBI GenBank NM_ pegfp- SLC26A4 HEK293 HEK293 2A 2B Western Blot 1 1 DL2000 Marker 2 pegfp-slc26a4 2 pegfp-slc26a4 HEK293 RT-PCR Western Blot a. HEK293 ( DAPI ); b. pegfp-slc26a4 ; c. RT-PCR : 1 DL5000 Marker 2 pegfp-slc26a4 cdna PCR bp β-actin; d. Western Blot : 1 87 kda 2 42~43kDa β-actin 2 a b 50 μm

4 80 Chinese Journal of Otology Vol. 9 No RT-PCR mrna (15 / 98) Belguith-Maalej [10] 52 2C 2D ( 22 (Graves' disease) 87 kda 3 Pendred Pendred 1896 Pendrin Everett [3] 1996 (autoimmune thyroid diseases) SLC26A Griffith [4] SLC26A4 Pendrin (LVAS) 1999 Abe [5] LVAS 7q31 Usami [6] SLC26A4 (Pendred ) SLC26A4 LVAS SLC26A4 7q [7] 0 ~ % (85 / 130); 23 ; 13 2 PCR SLC26A4 : 42 SLC26A %(42 / 130); 6 GJB2 Pendrin 4.6% (6 / 130); 36.9% pegfp-slc26a4 HEK293 (SLC26A4 GJB2 ) HEK293 [8] kda pegfp-slc26a4 S7-2A>G 83 S7-2 A>G 114 S7-2A>G S7-2A>G 12.69%(197 / 1552) 114 S7-2A>G PCR : (1) ; (2) PCR ; 1552 S7-2A>G (3) PCR S7-2A>G PCR % (161 / 1552) 78 SLC26A4 S7- pegfp-n1 2A>G ; 21 S7-2 A>G EGFP Kozak [12] 2% [9] 98 pegfp-slc26a4 EGFP N GJB2 29.6% (29 / 98) mtdna A1555G 4.08%(4 / 98) SLC26A % ) SLC26a4 Kim [11] Slc26a4 SLC26A4 PDS Pendrin Pendrin N C / 2A 2D Western Blot 87 RT-PCR SLC26A4 mrna bp SLC26A4 EGFP

5 HEK kda associated with enlarged vestibular aqueduct is caused by PDS mutations. Hum Genet (2): SLC26A (3): SLC26A4 S7-2 A>G (1): Yuan Y You Y Huang D et al. Comprehensive molecular etiology analysis of nonsyndromic hearing impairment from typical areas in China. J Transl Med : Everett LA Glaser B Beck JC et al. Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS). Nat Genet (4): Griffith AJ Arts A Downs C et al. Familial large vestibular aqueduct syndrome. Laryngoscope (8): Abe S Usami S Hoover DM et al. Fluctuating sensorineural hearing loss associated with enlarged vestibular aqueduct maps to 7q31 the region containing the Pendred gene. Am J Med Genet (4): Usami S Abe S Weston MD et al. Non -syndromic hearing loss (6): (5): Belguith -Maalej S Rebuffat SA Charfeddine I et al. SLC26A4 expression among autoimmune thyroid tissues. Immunobiology (5): Kim HM Wangemann P. Failure of fluid absorption in the endolymphatic sac initiates cochlear enlargement that leads to deafness in mice lacking pendrin expression. PLoS One (11): e CLONTECH Laboratories Inc. pegfp -N1 Vector Information [EB / OL]. ( ) [ ]. sisgene.com/vector/pegfp-n1.pdf ( : ) Richard Salvi

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