DNA. SPF 20 d 9. 09% % % % % % Testis Injection of Exogenous DNA Results in Expression in Mouse Sperm

Μέγεθος: px

Εμφάνιση ξεκινά από τη σελίδα:

Download "DNA. SPF 20 d 9. 09% % % % % % Testis Injection of Exogenous DNA Results in Expression in Mouse Sperm"

Ηιονη Αλεξιάδης
5 χρόνια πριν
Προβολές:

1 Chinese Journal of Zoology ~ 157 DNA * DNA Mus musculus DNA DNA pegfp-n 1 DNA- DNA μg / μl μg / μl μg / μl 3 SPF 2 5 d 3 20 d GFP PCR % % % 3 GFP PCR % % % DNA DNA μg / μl pegfp Q786 A Testis Injection of Exogenous DNA Results in Expression in Mouse Sperm XIE Chang YU Ming-Ju HUI Nan WANG Ling-Bin LI Wen-Jie ZHAO Yong-Ju * College of Animal Science and Technology Chongqing Key Laboratory of Forage & Herbivore Southwest University Chongqing China Abstract The aim of this study is to produce transgenic mice by testis-mediated gene transfer and investigate the suitable concentration of exogenous DNA for injection. A total of six SPF KM male mice were divided into three groups and liposome-treated exogenous DNA was directly injected into testis at three different concentrations μg / μl μg / μl and μg / μl respectively. Every treated mouse was naturally mated with 3 female mice on the 5th day after the injection. The spermatozoa were detected on the 20th day after injection under the fluorescence microscope and mouse testicular paraffin sections were prepared for detection of green fluorescent protein GFP expression under fluorescence microscope. The integration of pegfp-n 1 in the F1 was determined by PCR. The results showed that the proportion of florescent sperm in 3 groups were 9. 09% % and % respectively. Expression of GFP in different degrees was also found in three groups of paraffin sections. The PCR positive rates of offspring were % % and % respectively. It is concluded that the transgenic mouse could be produced by direct injection of foreign DNA into mouse testis and μg / μl was the suitable concentration of exogenous DNA for injection in this study. Key words pegfp Testis injection SMGT Transgenic mice No. 2008ZX No * zyongju@ 163. com xiechang_sd@ 163. com

2 152 Chinese Journal of Zoology 45 sperm-mediated gene transfer SMGT Marker DL2000 Plus DNA DNA- pegfp-n 1 SMGT DH5α 50 μg / ml LB pegfp-n ~ 2. 3 μg / μl μl μg 3 DNA 1 Lipofectamin Reagent DNA Kim ~ 6 mm 1999 Farre Sus domestica 47% 22% % F1 Mus musculus d Capra hircus 3 GFP 66. 7% ~ % F d 1 ml PBS 10 min 10 μl 488 nm DNA green fluorescent protein GFP SPF EDTA ph μl DNA μg ~ 98 DNA ph 9. 5 GFP DNA SPF 3 -CGTCGCCGATGGGTGGTGTTCT pegfp-n 1 Lipofectamine TM 507 bp 1. 5 μl DNA Invitrogen V gene F1 pegfp-n 1 PCR 5 ~ ~ 1. 0 cm - DNA pegfp-n ACGGCCACAAGTTCAGC-3 μl PCR 1. 5 μl DNA 2. 5 μl

2 DNA 153 10 buffer 15 mmol / L MgCl 2 1 μl 10 pmol / L 2 μl 2. 5 mmol / L dntp 0. 5 μl Taq 5 U / μl 25 μl PCR 94 5 min 94 45 s 60 45 s 72 75 s 35 2. 1 GFP 72 10 min 4 70 V 45 min SPSS for Windows 13.

3 2 DNA buffer 15 mmol / L MgCl 2 1 μl 10 pmol / L 2 μl 2. 5 mmol / L dntp 0. 5 μl Taq 5 U / μl 25 μl PCR 94 5 min s s s GFP min 4 70 V 45 min SPSS for Windows PCR % % % 1 Fig Spermatozoa transfected with pegfp-n1 under light- and fluorescence-microscope at 20 d A B C D A Light microscope Test group B Fluorescence microscope Test group C Light microscope Control group D Fluorescence microscope Control group GFP 3 3 F1 PCR F1 F1 4 1 ~ 6 DNA 500 bp 507 bp

Fig. 3 3 F1 The photo of F1 offspring under ultraviolet light 2.

4 154 Chinese Journal of Zoology 45 2 Fig. 2 GFP 200 GFP expression in mouse testis A B A Control group B Test group. Fig. 3 3 F1 The photo of F1 offspring under ultraviolet light 2. 4 DNA 7 d 1 Kim 3 Muramastu 8 Yamazaki EGFP SPSS for Windows F1 Yamazaki 9 PCR pegfp-n Yamazaki 9 10 d 3. 1

2 DNA 155 Fig. 4 4 F1 PCR The results of genomic DNA PCR test M. DL2000 Plus DNA C. 1 2 4 5 6 PCR 3 PCR M DL2000 Plus DNA marker C Control group 1 2 4 5 6 Positive PCR product 3 Negative PCR product.

5 2 DNA 155 Fig. 4 4 F1 PCR The results of genomic DNA PCR test M. DL2000 Plus DNA C PCR 3 PCR M DL2000 Plus DNA marker C Control group Positive PCR product 3 Negative PCR product. 1 Table 1 The experimental design and results Low concentration group Middle concentration group High concentration group Control group Injection dose μl Plasmid μg Liposome volume μl No. of male mice No. of female mice No. of pregnant F1 No. of offspring PCR No. of PCR positive PCR Rate of PCR positive % Yamazaki tpa PCR Farre 4 47% 133 Southern blotting 15 22% 14 Sato 10 PLNCXHCF Oryctolagus cuniculus F0 F2 85% 11 /13 Yonezawa 11 Rattus norvegicus 80% EGFP PCR Southern blotting Huang 12 intracytoplasmic sperm injection ICSI 15 4

6 156 Chinese Journal of Zoology 45 DNA μl DNA μg 3 1 DNA μg / μl μg / μl μg / μl μg / μl DNA PCR % DNA Sciamanna DNA DNA DNA DNA DNA 500 ng /10 6 DNA % DNA 17 75% 3. 3 DNA SMGT DNA DNA DNA inhibitory factor IF-1 20 d - DNA DNA DNA 20 Gallus gallus 23 Nakinishi 24 Lipofectamine 2000 DNA DNA 3. 2 DNA 10% ~ 15% Nakinishi 24 DNA DNA DNA Brachiller Rottman 22 DNA 3 DNA DNA- DNA DNA

7 2 DNA 157 DNA Villuendas 20 Kim 3 DNA DNA DNA PCR F1 1 Wall R J. New gene transfer methods. Theriogenology Yamazaki Y Fujimoto H Ando H et al. In vivo gene Kim J H Jung-Ha H S Lee H T et al. Development of a positive method for male stem cell-mediated gene transfer in mouse and pig. Mol Reprod Dev transfer to mouse spermatogenic cells by deoxy ribonucleic acid injection into seminiferous tubules and subsequent electroporation. Biology of Reproduction Cao Y Li Q Yuan X et al. Cloning of human lactoferritin 4 Farre L Rigau T Mogas T et al. Adenovirus-mediated gene and construction of expression vector. Journal introduction of DNA into pig sperm and offspring. Mol of Dalian Unversity of Technology Reprod Dev Villuendas G Gurierrez A Jimenez A et al. 1MV-driven expression of green fluorescent protein in male germ cells of transgenic mice and its effect on fertility. International 6. pegfp-n 1 Journal of Andrology Brachiller D Schellander K Peli J. Liposome-mediated DNA uptake by sperm cells. Mol Reprod Dev hcd Muramatsu T Shibata O Ryoki S et al. Foreign gene expression in the mouse testis by localized in vivo gene transfer. Biochem Biophys Res Commun Yamazaki Y Yagi T Ozaki T et al. In vivo gene transfer to mouse spermatogenic cells using green fluorescent protein as a marker. Exp Zool Sato M. Testis-mediated gene transfer TMGT in mice successful transmission of introduced DNA from F0 to F2 generations. Transgenics Yonezawa T Furuhata Y Hirabayashi K et al. Detection of transgene in progeny at different development stages following testis-mediated gene transfer. Mol Rerod Dev Huang Z Tamura M Sakurai T et al. In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially loclizaed jellyfish fluorescent protein gene. FEBS Letters Simth K R. Sperm cell mediated transgenesis a review. Anim Biotechnology Sciamanna I Piccoli S Barberi L et al. DNA dose and sequence dependence in sperm-mediated gene transfer. Mol Reprod Dev Rottmann O J Antes R Hofer P et al. Liposome mediated gene transfer via spermatozoa into avian egg cells. J Anim Breed Genet DNA Nakanishi A Iritani A. Gene treansfer in the chicken by sperm-mediated methods. Mol Repord

Σχετικά έγγραφα

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

, SLC26A4 HEK 293. The construction of wild-type SLC26A4 gene expression vector and the expression in HEK293 cells 2011 9 1 77 SLC26A4 HEK 293 1 2 1 1 1 1 (100853) 2 ( 050051) SLC26A4 (pegfp enhanced green fluorescent protein) SLC26A4 pegfp-slc26a4 HEK293 pegfp-slc26a4 HEK293 - (RT-PCR) SLC26A4 mrna Western Blot (