To harness the great capability of producing biologically active recombinant proteins with animal mammary glands, active research has been carried out in the past several decades to develop transgenic animals as bioreactors. However, when a transgene is introduced in the animal genome by random integration, the transgene tends to be subjected to epigenetic silencing, due to the so-called position effect from the chromatin environments surrounding the transgene integration sites, thereby resulting in low-level expression or total suppression. We report a universal transgenic strategy to knock in (KI) transgenes into the bovine β-casein gene locus allowing the expression of a transgene to be totally under the control of the endogenous regulatory sequences of the bovine β-casein gene. This universal KI strategy comprises two key components: one is the design of transcription activator-like effector nuclease (TALEN) constructs targeting the start codon region of bovine β-casein gene, and the other is the design of KI vectors in which a transgene of choice is flanked with homologous arms isolated from the ~500-bp bovine genomic DNAs immediately 5′ and 3′, respectively, of the translation start codon of the bovine β-casein gene. By using the human erythropoietin (hEPO) as the model transgene, we demonstrated that a transgene can be highly efficiently integrated immediately after the translation start codon of the bovine β-casein gene. In brief, the TALEN constructs were assembled by using the Golden Gate protocol. To KI the hEPO transgene, early passage (<5) of fibroblasts established from Holstein dairy cattle were cultured into full confluence in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), harvested with 0.25% trypsin-EDTA, and co-transfected with KI vector and the TALEN constructs by the Amaxa Nucleofector system. For each experiment, 106 cells were transfected with 5 μg of KI vector and 5 μg of TALEN constructs. After 72 h post-transfection, cells were harvested and subjected to limiting dilution to obtain single-cell derived colonies. To screen for single-cell derived colonies carrying the correctly KI of hEPO in the β-casein locus, we performed genomic PCR amplifying the genomic junctions created by the KI of hEPO gene into the bovine genome. We identified and established 2 hEPO transgenic bovine fibroblast cell lines after screening 10 single-cell derived colonies from the transfected cells (20%). The genotype of these 2 colonies was also confirmed by sequencing the PCR products. We have initiated the effort to produce hEPO transgenic cattle by somatic cell nuclear transfer (SCNT), and the animal cloning results will be reported at the conference.
Conformational rearrangements upon start codon recognition in human 48S translation initiation complex
