Abstract
To improve nutrient content of goat milk, we describe the construction of a vector (pBLAC) containing a hybrid goat β-lactoglobulin (BLG) promoter/cytomegalovirus (CMV) enhancer. We also describe the generation of transgenic goats expressing rhLA by somatic cell nuclear transfer (SCNT). Of 334 one-cell stage embryos derived from three transgenic cell lines and 99 embryos derived from non-transgenic (NT) cells surgically transferred to the oviducts of 37 recipients, two recipients delivered two kids (2%) from the non-transfected line and five recipients delivered six kids (1.8%) from transgenic cell lines, three of which died within 2 days. Compared to the NT donor cells, transfection of donor cells does not negatively affect the development of nuclear transfer embryos into viable transgenic offspring. However, the clone efficiency in cell line number 1 was lower than that in numbers 2 and 3, and in the NT lines (0.9% versus 1.9% 2.4% and 2%; P < 0.05). Two transgenic cloned goats expressed rhLA in the milk at 0.1–0.9 mg/mL. The mammary gland-specific expression vector pBLAC with hybrid BLG/CMV can drive the hLA gene to express in vitro and in vivo. These data establish the basis for use of a hybrid promoter/enhancer strategy to produce rhLA transgenic goats.
Highlights
The mammary gland is currently the only readily available animal bioreactor, but optimized expression vector construction is required for transgenic expression of recombinant proteins, and expression vectors still have strides to make [1, 2]
In our previous studies [7], a hybrid milk protein promoter (goat β-casein, bovine αs1-casein, and goat β-lactoglobulin (BLG)) with cytomegalovirus (CMV) enhancer was constructed and the expression level of recombinant human lactoferrin in the milk of transgenic mice was up to 8.2 mg/mL, which was more effective than using a single milk protein promoter (7– 40 ng/mL)
The expression plasmid containing the hLA genomic DNA (gDNA) was fused to the CMV sequence and flanked by the untranslated regions and flanking sequences of goat BLG gene, which contained 4.2 kb of 5 and 1.2 kb of 3 caprine BLG flanking no-coding sequence fused to 2.36 kb of hLA gDNA with a SV40 pA (Figure 1)
Summary
The mammary gland is currently the only readily available animal bioreactor, but optimized expression vector construction is required for transgenic expression of recombinant proteins, and expression vectors still have strides to make [1, 2]. Construction of the targeting vector is one of the most important techniques for mammary gland bioreactors, but the currently available techniques are laborious, time consuming, and inefficient [3]. In our previous studies [7], a hybrid milk protein promoter (goat β-casein, bovine αs1-casein, and goat β-lactoglobulin (BLG)) with cytomegalovirus (CMV) enhancer was constructed and the expression level of recombinant human lactoferrin in the milk of transgenic mice was up to 8.2 mg/mL, which was more effective than using a single milk protein promoter (7– 40 ng/mL). Construction of a hybrid milk protein promoter with enhancer is an attractive way to improve the expression level of recombinant proteins. The use of a hybrid promoter/enhancer is a valuable strategy in a transgenic mouse model, it is still unknown whether it is effective for other animals, such as goats
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