Human Lactoferrin (hlf) Cdna From Cells Expressing And Sperm-mediated Transfer Of Genetically Modified Animal Preparation

The plasmid pLNCXHLF which is combining with 2366bp human lactoferritin (hLF) cDNA complete sequence and 800bp regulating sequence upsteam by the β -lactoglobulin gene promoter is prepared by normal steps. Then it is used in the following experiments.The recombinant plasmids pLNCXHLF were introduced into mouse mammary tumor cell line MA-782 by liposome entrapment. Positive single clonal cells were selected with G418 and PCR. After proliferating, the transfected cells immobilized and cultured in soldium alginate were induced by hormone. The cultured cell suspensions tested by Western-blotting showed that transfected cells could express the exogenous gene and secrete human lactoferrin protein, with MW of 34 KD. The highest amount detected with ELISA reached 65mg/L medium / 105 cells. The recombinant hLF protein has the effect of inhibiting E.coli proliferation, whose activity is 1.4-1.8 times higher than the commercially available hLF.The recombinant plasmids pLNCXHLF were directly injected into male rabbit's testis and male goat's spermaductus. Using F1/R1 PCR system and southern hybridization, the transgenic positive rate of rabbit offspring genomic DNA is 35%(11/31). From the PCR result of Fl/Rl and F2/R2 system, the positive rate of goat offspring genomic DNA is 33.3% (4/12)and 25%(3/12) respectively. And the positive one is a female. Then, we prepared genome DNA of death goat offsprings from tongue, lung, liver, musule, skin, brain, germ gland, spleen, intestines, heart, and kidney. The result of Fl/Rl and F2/R2 system of PCR indicates that different tissues uptake extogenous DNA differently. Different amount of copies in different tissues attribute to the different density of positive signals. The result of the experiment suggested that the transgenic animals can be produced by spermatozoa-mediated gene transfer after the entrapment of liposome. And because the exogenous DNA occurs losing the segments .partly integration ,or existing outside of genome DNA , the rate of chimerism is relatively high.