Studies On Transgenic Mice And Cow With Lysine-rich Protein Milk

The inability of humans and many farm animals to synthesize certain amino acidshas triggered tremendous interest in increasing the levels of these so-called essentialamino acids in the human diet. Among the essential amino acids, lysine has receivedthe most attention because it is the most limiting amino acid in cereal crops, whichrepresent a major source of human food and animal feed worldwide. The lysine isconsidered the most important of the essential amino acids. In this study, milk proteincDNA clone fragments encoding lysine-rich proteins were spliced together togenerate a lysine-rich (LR) gene and the expression vector pBC1-LR-NEO,pcDNA3.1-LR-his and pcDNA3.1-LR were constructed. The effecy of LR gene andvector were identified by transient expression of cells transfection in vitro and cowmammary glands injection in vivo. Transgenic mice were generated by pronuclearmicroinjection of the linearized expression vectors harboring the LR transgene. Thetransgenic mice and their offspring were examined using multiplex PCR, Southernblotting, RT-PCR, in situ hybridization and Western blotting techniques. The stabletransgenic cell lines were obtained and used as donor cells for nuclear transfer.Finally,4 transgenic cloned cow were obtained.The main results were as follows:1.β-casein,αS2-casein and siderophilin cDNA clone fragments encoding lysine-richproteins were spliced together to generate a lysine-rich (LR) gene. To examine theexpression of LR gene, a His-tag protein cDNA fragments were re-amplified on its 5'end, the expression vector pcDNA3.1-LR-his was constructed. 293T cells weretransfected with pcDNA3.1-LR and collected after 48 h. The LR protein expressionwas confirmed by His-tag protein anibody.2. The LR gene was cloned into the Xho I restriction site of the pBC1 expressionvector, and the generation of the plasmid pBC1-LR was confirmed by DNA sequencing. For the stable transfection of bovine fibroblasts, a NEOrselection marker(the neomycin resistance gene from pGK-NEO-bpa, driven by the phosphoglyceratekinase promoter, was inserted into the Not I restriction site of the pBC1 expressionvector. The creation of pBC1-LR-NEOrwas confirmed by DNA sequencing. Themammary gland specific expression vector pBC1-LR-NEOrwas injected by directlyinjecting a mammary gland to express lysine-rich (LR) protein in cow. With thismethod, lysine-rich (LR) protein were expressed successfylly. The level of lysineincreased from 1.66 mg/ml (before transfection) up to 3.64 mg/ml (after transfectionday 3) in the milk.3. Transgenic mice were generated by pronuclear microinjection of the linearizedexpression vectors harboring the LR transgene. The transgenic mice and theiroffspring were examined using multiplex PCR, Southern blotting, RT-PCR, in situhybridization and Western blotting techniques. Our results show: (1) The LR gene wassuccessfully integrated into the mouse genome and was transmitted stably through thegerm-line within 3 transgenic generations. (2) Tissue- and stage-specific LR geneexpression was restricted to the mammary gland, active alveoli of the transgenicfemale mice during lactation. (3) The LR protein were successfully expressed intransgenic milk and secretes in the whey. The expression level of the LR proteinvaried among the three transgenic lines. In addition, there was no significant declinein LR expression among F0,F1and F2generations of transgenic mice. (4) The lysinelevel of the two transgenic lines was significantly higher than that of non-transgeniccontrols (p<0.05). (5) The growth performance of transgenic pups was enhanced bydirectly feeding them the LR protein-enriched transgenic milk.4. Bovine fetal fibroblast was transfected by expression vectors harboring the LRtransgene. The stable transgenic cell lines from 3 bovine fetuses were obtained andused as donor cells for nuclear transfer. Finally,4 transgenic cloned cow wereobtained.