Human lactoferrin transgenic rabbits produced efficiently using dimethylsulfoxide–sperm-mediated gene transfer
Lan Li A , Wei Shen A B D , Lingjiang Min A , Huansheng Dong A , Yujiang Sun C and Qingjie Pan A DA Institute of Animal Reproduction, Development and Genetic Engineering, Department of Animal Sciences and Technology, Laiyang Agricultural University, Chengyang, Qingdao 266109, China.
B College of Life Sciences, Peking University, Beijing 100871, China.
C Institute of Dongying Agricultural Sciences, Dongying, China.
D Corresponding author. Email: shenwei427@yahoo.com.cn
Reproduction, Fertility and Development 18(6) 689-695 https://doi.org/10.1071/RD06001
Submitted: 1 January 2006 Accepted: 2 May 2006 Published: 10 July 2006
Abstract
Transgenic animal mammary gland bioreactors are used to produce recombinant proteins. However, it is difficult to validate whether these transgenic domestic animals are able to express the recombinant protein efficiently in their mammary glands before the birth of transgenic offspring. In the present study, a simple and efficient method was established to evaluate the functionality of animal mammary gland tissue-expressed cassettes. The gene transfer vector pGBC2LF was constructed, and the expression of human lactoferrin (LF) gene was controlled by the goat β-casein gene 5′ flanking sequence. To obtain the most efficient transfection, the influence of DNA concentration, dimethylsulfoxide (DMSO) concentration, and the ratio of linear-to-circular DNA required for associating DNA with spermatozoa were evaluated. Transfection of exogenous DNA into rabbit spermatozoa was found to be efficient using 30 μg mL–1 DNA, DMSO at a final concentration of 3%, and a 3 : 1 ratio of linear-to-circular DNA, with 29 of 85 (34.1%) in vitro-fertilised embryos being transgenic. Using DMSO–sperm-mediated gene transfer (DMSO-SMGT), 89 rabbit offspring were produced, with 46 of these (57.1%) being transgenic. As mammary gland bioreactor models, 17 of 21 (81%) transgenic female rabbits could express human LF protein in their glands. During lactation of the transgenic rabbits, the highest level of human LF protein expressed was 153 ± 31 μg mL–1, and the mean expression level in all of the transgenic rabbits was 103 ± 20 μg mL–1 in the third week, declining gradually after this time. Our results demonstrate that transgenic rabbits produced by DMSO–SMGT were able to express human LF protein in the correct tissue.
Extra keywords: goat β-casein gene, in vitro fertilisation, mammary gland bioreactor.
Acknowledgments
We thank Professors Guoqing Qin and Wenru Tian for critical reading of the manuscript, Xiuhua Yu for the care of experimental animals, and Drs Yuqiang Shi, Zhengtian Yang and Liyuan Tian for their help in the molecular experiments. Grant sponsor: the State 863 High Technology R&D Project of China, grant number 2002AA206621. Grant sponsor: the key fund for doctoral scientific research startup of Laiyang Agricultural University. Grant sponsor: the key programs for science and technology development of Shandong province, grant number J05K06.
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* L. Li and W. Shen contributed equally to this work.
