PubMed2004
An expression construct specified in mammary gland with double cistrons of human G-CSF gene and IRES-EGFP gene under control of ovine beta-lactoglobulin gene flanking sequence, has been constructed in two parts (named fragment I and fragment II) that share an overlapping region of 2.2 kb sequence. Two sites of loxp and lox2272 for homologous recombination were inserted into both flanking regions of G-CSF. The lengths of fragment I and fragment II are 5.9 kb and 5.6 kb, respectively. The whole length of the expression vector (beta-LG-hG-CSF-IRES-EGFP) is 9.3 kb. The two DNA fragments mixed with nuclear locating sequence DNA (NLS) fragment were coinjected into murine zygote cytoplasm. The resulting mice were analyzed for the transgene integration and expression. A total of 138 founders were born. 62.3% (86/138) of them was integrated with fragment I sequence, and 54.3% (75/138) of them contained fragment II, whereas 62 of them contained both fragment I and II, of whom 80. 6% (50/62) were integrated by the whole reconstituted gene construct (result of ex-chromosomal homologous recombination, ECR). By RT-PCR analysis, it was shown that 90% of the ECR mice (9/10) expressed human G-CSF gene and 100% expressed EGFP gene. EGFP expression was also detected by absorption spectrum scanning from 400 nm to 700 nm, and 50% (5/10) of the ECR mice expressed EGFP protein. The high frequency and accuracy of homologous recombination in murine zygotes reported here suggests that some large transgenes could be constructed by ECR pathway. This method does not need to construct a long complex DNA vector directly and to do nucleus-injection, and is easier than traditional method.