Chinese Journal of Zoology 2010 45 2 151 ~ 157 DNA * 400716 DNA Mus musculus DNA DNA pegfp-n 1 DNA- DNA 0. 08 μg / μl 0. 12 μg / μl 0. 24 μg / μl 3 SPF 2 5 d 3 20 d GFP PCR 3 9. 09% 47. 06% 27. 78% 3 GFP PCR 17. 26% 47. 61% 22. 11% DNA DNA 0. 12 μg / μl pegfp Q786 A 0250-3263 2010 02-151-07 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 400716 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 0. 08 μg / μl 0. 12 μg / μl and 0. 24 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% 47. 06% and 27. 78% respectively. Expression of GFP in different degrees was also found in three groups of paraffin sections. The PCR positive rates of offspring were 17. 26% 47. 61% and 22. 11% respectively. It is concluded that the transgenic mouse could be produced by direct injection of foreign DNA into mouse testis and 0. 12 μg / μl was the suitable concentration of exogenous DNA for injection in this study. Key words pegfp Testis injection SMGT Transgenic mice No. 2008ZX08008-004 No. 30600430 * E-mail zyongju@ 163. com E-mail xiechang_sd@ 163. com 2009-09-10 2009-12-28
152 Chinese Journal of Zoology 45 sperm-mediated gene transfer SMGT Marker DL2000 Plus DNA 1. 2 1 1. 2. 1 DNA- pegfp-n 1 SMGT DH5α 50 μg / ml LB pegfp-n 1 2. 1 ~ 2. 3 μg / μl μl μg 3 DNA 1 Lipofectamin Reagent 1 1. 2. 2 DNA 1-2 1997 Kim 3 1 5 ~ 6 mm 1999 Farre Sus domestica 47% 22% 4 2002 34. 8% 1. 2. 3 F1 Mus musculus 5 2005 5 d 2008 12 14 Capra hircus 3 GFP 66. 7% 6 2009 1 1 ~ 3 2006 4. 3% F1 7 1. 2. 4 20 d 1 ml PBS 10 min 10 μl 488 nm DNA green fluorescent protein GFP SPF EDTA ph μl DNA μg 3 1 9. 0 92 ~ 98 DNA ph 9. 5 GFP DNA 1 1. 1 1. 1. 1 SPF 3 -CGTCGCCGATGGGTGGTGTTCT-5 1. 1. 2 pegfp-n 1 Lipofectamine TM 507 bp 1. 5 μl DNA 25 2000 Invitrogen V gene 1. 2. 5 F1 pegfp-n 1 PCR 5 ~ 10 0. 5 ~ 1. 0 cm - DNA pegfp-n 1 5 - 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. 0 PCR 2 3 1. 3 9. 09% 47. 06% 27. 78% 1 Fig. 1 1 200 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. 2. 2 GFP 3 3 F1 PCR 1 2 4 5 6 2 3 3 2. 3 F1 F1 4 1 ~ 6 DNA 500 bp 507 bp
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 9 2. 5 EGFP SPSS for Windows 13. 0 F1 Yamazaki 9 PCR pegfp-n 1 2 1 3 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. 1 Table 1 The experimental design and results Low concentration group Middle concentration group High concentration group Control group Injection dose μl 50 50 50 50 Plasmid μg 4 6 12 0 Liposome volume μl 12 18 36 0 No. of male mice 2 2 2 2 No. of female mice 6 6 6 6 No. of pregnant 4 5 3 5 F1 No. of offspring 52 61 38 59 PCR No. of PCR positive 9 29 8 0 PCR Rate of PCR positive % 17. 26 47. 61 22. 11 0 Yamazaki 9 13 1 tpa 5 382 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
156 Chinese Journal of Zoology 45 DNA μl DNA μg 3 1 DNA 0. 08 μg / μl 0. 12 μg / μl 0. 24 μg / μl 0. 12 μg / μl DNA PCR 47. 61% DNA Sciamanna 16 17 DNA DNA DNA DNA DNA 500 ng /10 6 DNA 3 1 72% DNA 17 75% 3. 3 DNA SMGT DNA DNA DNA 18-19 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 21 1991 Rottman 22 DNA 3 DNA DNA- DNA DNA
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