The goat β‑casein/CMV chimeric promoter drives the expression of hLF in transgenic goats produced by cell transgene microinjection

Abstract

There is growing interest in the application of lactoferrin (LF) as a drug or food additive for animals and humans. The objective of this study was to produce transgenic cloned goats that would serve as living bioreactors, expressing high levels of recombinant human LF (rhLF) in their milk. We designed a pCL25 expression vector containing goat β-casein/CMV chimeric promoter in order to facilitate rhLF expression. This pCL25-rhLF-Neo vector was microinjected into goat fetal fibroblasts. G418 selection and PCR analysis were used to identify transgenic donor cells suitable for somatic cell nuclear transfer (SCNT). After SCNT and embryo transplantation, goats harboring the hLF gene were produced, as confirmed via PCR and southern blotting. The average rhLF concentration in milk from this transgenic goat was 3.89 mg/ml as determined via ELISA. We also used an optimized buffer in order to effectively elute high-purity (95.8%) rhLF from a cation-exchange column, with the recovered rhLF exhibiting high biological activity. Findings from this study demonstrated that it is possible to generate a transgenic goat harboring the hLF transgene driven by the goat β-casein/CMV chimeric promoter. It represents an initial step towards the production of rhLF, potentially allowing for industrialized purification in the future.

Generating Cloned Goats by Somatic Cell Nuclear Transfer—Molecular Determinants and Application to Transgenics and Biomedicine

“…The nuclear transfer of in vitro-transfected somatic cells increases the probability of producing nonmosaic transgenic offspring, which have an exogenous gene construct incorporated into the primordial germ cell line. Such specimens, which are identified as nonchimeric with regard to the genetic transformation of gametogenic and somatic cells, retain their full capacity to transmit phenotypically and molecularly diagnosed transgene expression to the secretory epithelial cells (lactocytes) in the mammary glands of the next generation of kids [ 49 , 55 , 56 ]. An outstanding example is found in the findings of Baguisi et al [ 45 ].…”

“…Diminishing the capability of the milk provided by genetically engineered bioreactors to trigger acute allergic reactions in humans is simultaneously reflected in enriching the goat milk with the valuable multipotent protein designated as LF. This immune glycoprotein, apart from physiologically regulating the dynamic homeostasis of the metabolism of iron cations, is characterized by several other desirable immunotherapeutic properties, including antimicrobial (antibacterial, mycostatic, and antiviral), immunomodulatory, anti-inflammatory, and anticancer abilities ( Table 1 ) [ 22 , 24 , 56 ].…”