Using layered double hidroxides in ovine sperm-mediated gene transfer


BAKIRER ÖZTÜRK G., GÖK M. K. , DEMİR K., ARICI R., Can A., Ertürk E., ...More

12th Transgenic Technology Meeting (TT2014), Edinburg, United Kingdom, 3 - 06 October 2014, vol.23, no.5, pp.858

  • Publication Type: Conference Paper / Full Text
  • Volume: 23
  • City: Edinburg
  • Country: United Kingdom
  • Page Numbers: pp.858
  • Istanbul University-Cerrahpasa Affiliated: No

Abstract

Layered double hidroxides (LDH) are chemical materials that can be synthesized from many different substances, have double lipid layers in their strucutres and it is highly important to determine their sizes, working pH values and synthesis methods in order to use them as transfection agents. When DNA molecules are being kept in between double lipid layers, it is aimed that it releases the DNA molecule in order to achieve a successful transfection in cells. LDH’s are used in some cell transfection researches but they have not been used as a new vehicle in sperm-mediated gene transfer (SMGT) yet.

In our study, we used oocytes obtained from ovaries of slaughtered sheeps and used two different kins of LDH’s Mg- Al-Cl_LDH and Ca-Al-Cl-LDH with the concentration of 2.5 ng/lL. pDNA (plasmid DNA) for the transfection of ram spermatozoa LDH’s were mixed with DNA at 37oC for 4 h, after that LDH-DNA complexes were incubated with spermatozoa for 1 h at room temperature. ICSI (intracytoplasmic sperm injection) was used to fertilize the MI ovine oocytes. We used EGFP (enhanced green fluorescent) gene to determine transgenesis under attempted with transfection. The controls of transgenic embryos are achieved according to the green luminescence status under fluorescent attached invert microscobe. All the embryonic development procedures were made as in Birler et al. 2010, except we used only ionomycine activation and put oocytes in fertilization SOF media after ICSI.

Our results showed that in both Mg-Al-Cl LDH and Ca-Al- Cl-LDH groups we obtained had transgenic embryos 13.04 % and 21.87 %, respectively, but not statistically important (p[0.05). The cleavage ratios were 74.19 % and 71.11 % also the degeneration rates were 25.80 and 26.66, respectively, but not statistically important (p[0.05). The embryonic development ratios of the LDH groups were not lower than our control group. In conclusion, we determined that both Mg-Al-Cl_LDH and Ca-Al-Cl-LDH can be used for SMGT in ovine transgenesis. They did not affect embryonic development negatively, and their transgenesis efficiency can be elevated with more researches in this field.