The DNA was transferred onto a positively charged nylon membrane (Roche) and hybridized with the DIG-labeled hTPO cDNA fragments.
The first PCR primers for the hTPO cDNA were as follows: 5'-ggagctgactgaattgctcctcgt-3' and 5'-cctgacgcagagggtggaccctcc-3'.
We constructed two knock-in vectors to direct expression of the hTPO gene under control of the endogenous beta-casein promoter at the bovine beta-casein locus (Figure 1A and B).
clones were further confirmed by Southern blot analysis; a specific 9.9-kb signal band was detected after hybridization using a probe from the hTPO cDNA (Figure 2E).
Transgenes of cloned embryos were analyzed at various developmental stages by PCR amplification for hTPO. Twenty-three (88%) of 26 cloned embryos derived from KI-81 cells and 43 (83%) of 52 from KI-89 cells were positive for the transgene (Figure 3A and Table 2).
In this report, we successfully achieved, for the first time, knock-in of the hTPO gene on the beta-casein locus of bovine fibroblasts.