To Establish The Techniques For Nucleofectoring Mouse Spermatozoa In Vitro And Investigate The Primary Mechanism

Transgenic techniques have rapidly evolved in recent years. There are five strategies including more than a dozen of experiment protocols available to generate genetically modified animals. However, the efficiency of these techniques to produce viable offspring is still disappointingly low. Pronuclear microinjection of DNA constructs continues to be the most widespread method of transgenic animal production; however, the procedure is limited in its applications and remains very costly. Microinjection requires a large number of fertilized embryos due to the high mortality rate. Pronuclear visualization is necessary and requires significant embryo manipulation, including centrifugation, in many domestic animal species. Skilled technicians are required, due to the highly technical nature of the procedure, and even in the hands of these highly skilled technicians, the efficiency of the procedure and in vitro culturing techniques remains unsatisfactory. People are always to look for an alternative strategy to pronuclear microinjection. Sperm-mediated gene transfer (SMGT) which uses sperm cells interact with exogenous DNA as vectors for DNA delivery during fertilization to generate transgenic animals, is one of the most desirable methods used to achieve transgenesis as an alternative to pronuclear microinjection, because it is simple, convenient, commonly used that nearly adapt allkind of species in methodology. In 1989, Lavitrano et al. reported utilizing SMGT to achieve transgenic mice, sparking wide spread excitement in the scientific community. During the last decade, a number of different approaches for making transgenic spermatozoa have been developed and the mechanism of interact of spermatozoa with DNA also have been elucidation. However, the efficiency and reliability of SMGT are still controversial, except with the method of Intracytoplasmic sperm injection (ICSI) developed by Perry AC in 1999. Collecting the available data about SMGT and analyzing the all kind of phenomena, we gained the conclusion that it is the destination for SMGT to enhance the efficiency in nucleofector. For this destination, we design the experiment program based on the widely used gene transfer techniques for quiescent or weakly dividing cells, one is rectangle pulse electroporation, the other is a new type synthetic DNA delivery, polyethylenimine (PEI). The two gene delivery methods are believed to act by different mechanisms. This study was designed to determine if genes packaged using PEI might be more effectively delivered into sperm nucleus and at the same time low cell mortality in combination with electroporation. We carried out three experiment in series: (DThe component design of electroporation media for spermatozoa. (2) Determin the eletric pulse parameters in condition that using sperm electroporation media.(3)Gene packed using PEI in combination with electroporation facilitate the exogenous DNA entry into the nucleus of mouse sperm.All the experiments are divided into three parts. Part 1: The component design of electroporation media for spermatozoa.Electroporation is an efficiency gene transfer strategy for all kinds of cells, but the procedure is limited in sperm-mediated gene transfer (SMGT) because of lacking special electroporation buffer designed for spermatozoa. Referencing many prescription of electroporation buffer used in eminent laboratory and analyzing theprinciple of electroporation, we summarize the guide for component design of electroporation buffer. The rule was described as low sodium, high potassium, low calcium, high magnesium, low conductivity and supplement osmotic pressure with non-electrolyte. We divided the buffer component into groups in order to lower the difficulty of the experiment. We observed the change of sperm morphous in buffer with difference composition. We take the speed that sperm swell and deform in electroporation buffer and the recovery after replacing with cell culture media as a criterion for adaptability to sperm. We also observed the late effect of electroporation buffer, comparing the vitality and motility after going through the procedure that sperm culture in electroporation buffer for 2 hours, following replacing with cell culture media for 6 hours. Our conclusion: (1) Compared with the traditional electroporation buffer composition, the buffer solution with a lower concentration of potassium ions and a higher concentration of sodium is more suitable for sperm.(2) Too high concentration of magnesium ions will aggravate damage caused by the solution with low sodium, high potassium and bring about the decrease in vitality and motility of sperm. (3)It is advantage for sperm to supplement appropriate glycerol into electroporation buffer solution. The final composition of electroporation buffer for sperm is NaCl 50 mM, KCllOmM, MgSO4 2 mM, Na2HPO4 8.1 mM , KH2PO4 1.47 mM, Pyruvate 0.33 mM, Glutathion 5 mM, Glucose 140 mM, Glycerol 1%.Part 2: Determine the electric pulse parameters in condition that using sperm electroporation media.A new type rectangle pulse electroporation system is capable to easily set pulse width, amplitude, pulse number and time between pulse, as well as set pulse groups according to different destination. Comparing with traditional electroporation systemusing exponential decay pulser, rectangle pulse electroporation is lower cell damage, higher efficiency of gene delivery and more repeatable, which provid a new chance for sperm-mediated gene transfer. Using viability and motility as evaluation criterion, we determine approximately the electric pulse parameters. We determine the exact parameters by observing the uptake of propidium iodide and viability of mouse sperm after dealing with electroporation. Finally, we use the FITC-conjugated double-stranded oligonucleotides as a macromolecule electroporation model to detect the experimental parameters determined by observing the uptake of propidium iodide. In this experimental condition(groupl: 2 pulse amplitude=360V, width=65 u s, interval=0.75s; group2: 2 pulse amplitude=90V, width=5ms, interval=ls), the mouse sperm have appropriate molecular uptake and cell viability.Part 3: Gene packed using PEI in combination with electroporation facilitate the exogenous DNA entry into the nucleus of mouse sperm.The one of major factors that the efficiency and reliability of Sperm-mediated gene transfer (SMGT) to produce transgenic animals is still disappointingly low is difficult to delivered exogenous DNA into sperm nucleus. To develop improved methods of SMGT, packaging DNA in synthetic DNA delivery vectors polyethylenimine (PEI) in combination with electroporation can facilitate the exogenous DNA entry into the nucleus of mouse sperm. To test this hypothesis, we used cationic polymer PEI labelled by fluorescein isothiocyanate (JetPEI- FluoF) compound with plasmid DNA at appropriate ration to form JetPEI-FluoF-DNA complex. The complex can act as tracing material to show the degree of nucleofector in sperm when we observed the sperm with confocal laser scanning microscope. We use this complex in sperm electroporation at optimization pulse parameters. The result in contrast to the group using PEI alone without electroporation, the viabilityand motility of sperm were a little decrease but the fluorescein material was increased largely either in cytoplasm or in the cell nucleus area. We conclude that the combination of electroporation with PEI improve gene transfection for mouse sperm in vitro. The method described in this experiment might be a novel approach for SMGT.