Abstract
As an initial step towards creating genetically modified cattle as a biopharming source of recombinant human serum albumin (rHSA), we report modification of the bovine albumin (bA) locus by transcription activator-like effector nuclease (TALEN)-stimulated homology-directed repair (HDR). Pedigreed bovine fibroblasts were co-transfected with TALENs and an 11.5-kb human serum albumin (HSA) minigene donor construct, designed to simultaneously disrupt and replace bovine serum albumin (BSA) expression with controlled rHSA expression in both the liver and the milk. Targeted integration of the HSA minigene was confirmed in transfected fibroblasts at a frequency of approximately 11% and transgenic bovine embryos were produced from targeted fibroblasts using somatic cell nuclear transfer (SCNT). The research delineated here lays the foundation for the future generation of transgenic rHSA cattle with the potential to provide a large-scale, reliable, and quality-controlled source of rHSA.
Highlights
There is a vast clinical need for human serum albumin (HSA), with over 500 metric tons used worldwide every year [1,2]
Our approach is to humanize the endogenous bovine albumin gene, replacing it with a dually expressing HSA minigene construct, which will allow for normal expression of recombinant HSA (rHSA) protein in the liver, as well as its exogenous expression in the milk
We show that these targeted cells can be used as nuclear donors in somatic cell nuclear transfer (SCNT) and are capable of directing development to at least the blastocyst stage
Summary
There is a vast clinical need for human serum albumin (HSA), with over 500 metric tons used worldwide every year [1,2]. Previous bovine transgenics [3] which express rHSA in the milk have not proved commercially viable This is due to the transudation and transcytosis [5] of endogenous bovine serum albumin (BSA), a highly conserved ortholog of HSA, into the milk [6], necessitating an expensive and tedious purification process. Our approach is to humanize the endogenous bovine albumin (bA) gene, replacing it with a dually expressing HSA minigene construct, which will allow for normal expression of rHSA protein in the liver, as well as its exogenous expression in the milk. Such cattle can potentially provide an economically feasible alternative for a safer and more reliable source of therapeutic rHSA
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