Effects of Sperm Pretreatment on Efficiency of ICSI-Mediated Gene Transfer in Pigs

Abstract

Abstract. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer has recently been shown to be an effective technique for producing transgenic pigs; however, the types of sperm pretreatment having the most beneficial effects on post-ICSI embryogenesis or transgenic efficiency have not been clarified. In the present study, we performed ICSI-mediated gene transfer using pig sperm subjected to various pretreatments and determined the developmental potential of sperm-injected oocytes and introduction efficiency of exogenous DNA. Embryos were then transferred to recipient pigs to confirm gene transfer efficiency during the fetal period. When ICSI was performed using unfrozen sperm heads with tails removed by piezo-pulse, the rates of blastocyst formation (14.2%, 17/120) and transgene (EGFP) expression (11.8%, 2/17) were both low. When unfrozen sperm heads were used that were removed by sonication, EGFP expression efficiency (11/21, 52.4%) improved significantly (P<0.05). Pretreatment of unfrozen sperm with a surfactant or acrosomal reaction did not further improve the rates of blastocyst formation and EGFP expression. However, use of the heads of sperm frozen-thawed with or without a cryoprotective agent resulted in rates of blastocyst formation and EGFP expression that tended to be generally high (23.0%, 14/61-33.8%, 26/77 and 42.9%, 6/14-66.7%, 10/15). A total of 219 in vitro matured oocytes were fertilized by ICSI-mediated gene transfer using the heads of frozen-thawed sperm and then transferred into two recipient pigs. Seven fetuses were obtained, and EGFP expression and integration of the transgene (10-30 copies) were confirmed in two of the seven fetuses. Use of unfrozen sperm thus confers no advantages on ICSI-mediated gene transfer, and although further investigations are needed, frozen-thawed sperm heads appear to be useful in ICSI-mediated gene transfer.

Efficiency of Sperm-Mediated Gene Transfer in the Ovine by Laparoscopic Insemination, In Vitro Fertilization and ICSI

“…In mice and pigs, there has been no discussion regarding the state of the transgene after transgenic ICSI. In both species, it has been demonstrated that 5-25 transgene copies are integrated in one or two host genome loci [11,17]. Unfortunately, the size of transgene (5.5 kb) and amount of DNA in a blastocyst (<6 pg) make it very difficult to confirm transgene integration through techniques such as FISH or Southern blot in our experience.…”

“…However, results obtained so far in mice and domestic animals have been inconsistent [10,[12][13][14][15][16]. On the other hand, ICSI has resulted in the production of transgenic offspring in murine and porcine species, while Southern Blot and FISH analyses in resultant embryos and offspring have proven efficient in demonstrating successful gene integration [11,17]. However, for successful large-scale application of transgenic ICSI to domestic species, it is critical to solve the ICSI fertilization problem associated with activation failure [18].…”

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

“…In addition to PNI and SCNT, lentiviral transduction of zygotes 24,25 and intracytoplasmatic sperm injection (ICSI) 26,27 have been employed for transgenesis in the pig. Lentiviral transduction can produce high ratios of transgenic offspring (20-30 % transgenic offspring per treated embryos), but this technique frequently results in cell mosaicism and thus reduced germline transmission; the maximal cargo of foreign DNA with lentiviral vectors is about 7 kbp; and epigenetic silencing of the virally integrated constructs has been observed 13 .…”

“…Another limitation is that a laboratory with biosafety level 2 is required for lentivirus production and handling. ICSI of membrane-damaged (dead) spermatozoa incubated with foreign DNA has been used to produce transgenic piglets; however, this method is technically demanding and the reported success rates are low (<1 % transgenic offspring per treated oocytes) 26 .…”

Transgenesis in farm animals-A review

“…A study showed that despite of obtaining high rates of morulae and blastocysts through IVF and LI, none of the embryos expressed the transgene (Perry et al,1999). On the other hand, various reports indicate that ICSI-mediated gene transfer is an effective and repeatable method to produce transgenic mice ( Perry et al, 1999) and pigs (Kurome et al, 2007). In 2011, by using ICSI, 91.6% GFP expressing embryos were obtained (Pereyra-Bonnet et al, 2011).…”