Untitled

Abstract

Production of transgenic livestock by pronuclear microinjection of DNA into fertilized zygotes suffers from the compounded inefficiencies of low embryo survival and low integration frequencies of the injected DNA into the genome. These inefficiencies are one of the major obstacles to the large-scale use of pronuclear microinjection techniques in livestock. We investigated exploiting the properties of recombinase proteins that allow them to bind DNA to generate transgenic animals via pronuclear microinjection. In theory, the use of recombinase proteins has the potential to generate transgenic animals with targeted changes, but in practice we found that the use of RecA recombinase-coated DNA increases the efficiency of transgenic livestock production. The use of RecA protein resulted in a significant increase in both embryo survival rates and transgene integration frequencies. Embryo survival rates were doubled in goats, and transgene integration was 11-fold higher in goats and three-fold higher in pigs when RecA protein-coated DNA was used compared with conventional DNA constructs without RecA protein coating. However, a large number of the transgenic founders generated with RecA protein-coated DNA were mosaic. The RecA protein coating of DNA is straightforward and can be applied to any species and any existing microinjection apparatus. These findings represent significant improvements on standard pronuclear microinjection methods by enabling the more efficient production of transgenic livestock.

No Effect of Recombinase-Mediated DNA Transfer on Production Efficiency of Transgenic Rats

“…The protective action of RecA-coating agreed well with the results of Maga et al [11]. However, the offspring rate from oocytes injected with the ssEGFP/ RecA (20%) was comparable with that from those injected with dsEGFP (26%), when non-cleaved as well as cleaved oocytes were transferred into the recipients.…”

“…Three out of five (60%) PCR-positive offspring derived from the recombinase-mediated DNA transfer method were the green rats. A high incidence of mosaicism has been reported in transgenic goats and pigs produced by RecA-coated DNA injection [11]. The true mosaicism of founder rats as assessed by transmission of the transgene to progeny was not investigated in the present study, and because of the small sample number, it remains to be clarified whether integration of transgenes into rat genomes is likely to be mosaic.…”

“…Another approach to production of transgenic animals is intracytoplasmic sperm injection (ICSI)-mediated transgenesis (ICSI-Tg) [16], in which <7% of inseminated oocytes develop into transgenic mice [16,20] and rats [7,9]. Maga et al [11] recently reported that recombinase-A protein (RecA)-coated …”

“…Another approach to production of transgenic animals is intracytoplasmic sperm injection (ICSI)-mediated transgenesis (ICSI-Tg) [16], in which <7% of inseminated oocytes develop into transgenic mice [16,20] and rats [7,9]. Maga et al [11] recently reported that recombinase-A protein (RecA)-coated ssDNA was more likely to be integrated into goat and pig genomes after pronuclear microinjection (6-and 10-fold higher, respectively). Very recently, Kaneko et al [8] have also reported that the ICSI-Tg using RecAcoated ssDNA produced transgenic mice at efficiencies of 2.9-7.7% of inseminated oocytes, while ICSI-Tg using the same ssDNA without RecA-coating resulted in no transgenic offspring.…”

“…The recombinase protein, RecA, was bought from Roche Diagnostics (Basel, Switzerland). According to the method reported by Maga et al [11], linear dsEGFP DNA (100 ng) was heatdenatured at 98˚C for 5 min, and cooled on ice for 1 min. Then, the ssEGFP DNA was mixed at 4˚C with 4.2 µg RecA protein in 10 mM Tris-acetate buffer containing 2 mM magnesium acetate and 2.4 mM ATPγS.…”