Producing Transgenic Pig by Bacteria Magnetic Particles

doi:10.3969/j.issn.1674-5817.2015.04.005

Abstract

Abstract: Objective To establish sperm-mediated transgenic pigs models by intratestis injection with bacteria magnetic particles (BMP) and polyethylenimine (PEI) as a vector. Method The Lp-PLA2- GFP plasmid were mixed with BMP-PEI to BMP-PEI- PLA2-GFP, which were delivered into Bama Mini-pigs by intratestis injection. The sperm were collected in 2 weeks, 4 weeks, 6 weeks to 20 weeks after inoculation, and the GFP gene were detected by RT-PCR and immunofluorescence. The positive sperms will be transferred through artificial insemination to sows to establish transgenic pigs. The piglets were detected by RT-PCR to GFP gene and Real-time PCR to Lp-PLA2 gene. Results The GFP gene was first appearance in semen in 4 weeks after inoculation, and sustained through 16 weeks. The GFP protein mainly expressed in the sperm acrosome, sperm tail and neck area were observed by fluorescence microscopy. Through artificial insemination, 21 piglets were born, 9 of which were found with GFP segments, and the expression of PLA2 was obviously higher than that of other pigs, with a total positive rate of 42.86%. Conclusion The BMP combined with PEI can serve as a good non viral vector carrying exogenous gene, and sperm-mediated method with the vector can achieve the purpose of rapid preparation of transgenic pigs, its cost is low, the method is simple and convenient.

Key words: Pig, Transgenic, Bacteria magnetic particles (BMP), Polyethylenimine (PEI), Sperm-mediated gene transfer

CLC Number:

Q95-33

CHEN Ke-yan, CHEN Zhen-wen, DONG Wan-wei, ZHENG Zhi-hong. Producing Transgenic Pig by Bacteria Magnetic Particles[J]. Laboratory Animal and Comparative Medicine, 2015, 35(4): 288-293.

References

[1] Benam KH, Dauth S, Hassel B, et al.Engineered in vitro disease models[J] .Annu Rev Pathol, 2015, 10:195-262.
[2] Kvon EZ.Using transgenic reporter assays to functionally characterize enhancers in animals[J]. Genomics, 2015, 7543(15):30012-30014.
[3] Merkel OM, Urbanics R, Bedocs P, et al.In vitro and in vivo complement activation and related anaphylactic effects associated with polyethylenimine and polyethylenimine-实验动物与比较医学 Laboratory Animal and Comparative Medicine graft-poly block copolymers[J]. Biomaterials, 2011, 32(21):4936-4942.
[4] Colombo M, Carregal S, Casula MF, et al.Biological applications of magnetic nanoparticles[J]. Chem Soc Rev, 2012, 41(11):4306-4334.
[5] 吴艳花, 杜小燕, 陈振文, 等. 封闭群实验用小型猪遗传标准的建立[J]. 实验动物科学, 2010, 27(6):34-37.
[6] 袁进, 顾为望. 小型猪作为人类疾病动物模型在生物医学研究中的应用[J]. 动物医学进展, 2011, 32(2):108-111.
[7] 戴建军, 李翔, 吴彩凤, 等. 小鼠睾丸注射不同转染试剂和注射方法对外源基因表达的影响[J].生物工程学报, 2014,30(10):1522-1530.
[8] Yamazaki Y, Fujimoto H, Ando H.In vivo gene transfer to mouse spermatogenic cells by deoxyribonucleic acid injection into seminiferous tubules and subsequent electroporation[J]. Biol Reprod, 1998, 59(6):1439-1444.
[9] 阴彦辉, 孙敏, 陈庭锋, 等. 睾丸注射法制备携带山羊H-FABP基因的转基因小鼠[J]. 遗传, 2012, 34(6):727-35.
[10] Li T, Gendelman HE, Zhang G, et al.Magnetic resonance imaging of folic acid-coated magnetite nanoparticles reflects tissue biodistribution of long-acting antiretroviral therapy[J]. Int J Nanomedicine, 2015, 10(6):3779-3790.
[11] Takamura T, Ko PJ, Sharma J, et al.Magnetic-particle-sensing based diagnostic protocols and applications[J]. Sensors (Basel), 2015,15(6):12983-12998.
[12] Panagiotopoulos N, Duschka RL, Ahlborg M, et al.Magnetic particle imaging: current developments and future directions[J]. Int J Nanomedicine, 2015, 10(4):3097-3114.
[13] Watcharin W, Schmithals C, Pleli T, et al.Detection of hepatocellular carcinoma in transgenic mice by Gd-DTPA- and rhodamine 123-conjugated human serum albumin nanoparticles in T1 magnetic resonance imaging[J]. J Control Release, 2015, 199:63-71.
[14] Puhl MD, Mintzopoulos D, Jensen JE, et al.In vivo magnetic resonance studies reveal neuroanatomical and neurochemical abnormalities in the serine racemase knockout mouse model of schizophrenia[J]. Neurobiol Dis, 2014, 73C:269-274.
[15] 唐秋莎, 张东生, 顾宁, 等. 新型纳米基因载体(PEI/Mn_(0.5)Zn_(0.5)Fe_2O_4) 的制备、表征及体外实验[J]. 功能材料, 2007, 38(8):268-272.
[16] Sonawane ND, Szoka FC Jr, Verkman AS.Chloride accumulation and swelling in endosomes enhances DNA transferby polyamine-DNA polyplexes[J]. J Biol Chem, 2003, 278(45):44826-44831.