**3.5 Electroporation**

Electroporation or electropermeabilization has been used to transfer foreign plasmid DNA into bacteria, yeast, and mammalian cells (Escoffre et al., 2009; Favard et al., 2007; Golzio et al., 2010; Mir, 2009; Neumann et al., 1982; Somiari et al., 2000). It might be difficult to transfer large genomic DNA molecules into mammalian cells by this method. We might first digest the normal human genomic DNA by restriction enzymes, and then transfer the normal genomic DNA fragments into a patient's stem cells and somatic cells by electroporation in vitro. After a few days of in vitro cell culture in serum free media, the cells can be transplanted into the same patient. Of course, before conducting human clinical trials, this kind of experiment should be performed in animal models first.

#### **3.6 Genomic DNA Transfection**

Whole genomic DNA molecules are too big to be transfected into cells directly. Normal genomic DNA can be digested by a few restriction enzymes first, and then the purified genomic DNA fragments can be transfected into stem cells and somatic cells from patients.

Microinjection technique has been used in transgenic animals for many years (Bishop & Smith, 1989; Chan & Yang, 2009; Charreau et al., 1996; Filipiak & Saunders, 2006; Ménoret et al., 2010; Tesson et al., 2005; Yang et al., 2008). Microinjection technique also has been used as a tool to clone animals-first, an unfertilized egg's nucleus is removed; then a nucleus of a somatic cell is microinjected into the denucleated egg; now the egg contains a whole copy of the diploid genomic DNA from the somatic cell and can be cultured in vitro to form a blastocyst; and the blastocyst is implanted into the womb of an animal; eventually a cloned animal is born (Campbell et al., 1996; Vajta & Gjerris, 2006; Willadsen, 1986; Wilmut

Several different genes inserted into plasmids were microinjected into cultured mammalian somatic cells, and some genetic defective genes were corrected by homologous recombination (W. Anderson et al., 1980; Capecchi, 1980; Folger et al., 1982; Yamaizumi et

Feng et al. introduced a 110 kb whole human alpha globin gene cluster clone in a bacterial artificial chromosome (BAC) vector into fertilized eggs to generate transgenic mice by microinjection method. The human alpha globin gene cluster DNA was integrated into the mice genome, and human alpha globin mRNA was expressed in 3 transgenic mice (Feng et al., 2001). Similarly, Gao et al. generated transgenic mice carrying a BAC clone of a 116 kb human *apoAI/CIII/AIV/AV* gene cluster and a mutant in which the *apoCIII* enhancer was

I assume that normal genomic DNA without a plasmid or an artificial chromosome can be directly microinjected into the nucleus of somatic cells and stem cells from a patient successfully. This method could have a higher homologous recombination rate and less immunological reactions. It is not a very convenient method, but I think it is worth the effort to try. It only needs 30 purified mouse hematopoietic stem cells to save 50% of lethally irradiated mice (Spangrude et al., 1988). Even one single stem cell transplant can significantly reconstruct the bone marrow function of some irradiated mice (Decker & Nyberg, 2001; Krause et al., 2001; Mankad et al., 2006; Osawa et al., 1996). Therefore, we might need to collect less than one hundred stem cells from a patient, and microinject normal genomic DNA into these cells. Hopefully, less than one hundred of these corrected

Electroporation or electropermeabilization has been used to transfer foreign plasmid DNA into bacteria, yeast, and mammalian cells (Escoffre et al., 2009; Favard et al., 2007; Golzio et al., 2010; Mir, 2009; Neumann et al., 1982; Somiari et al., 2000). It might be difficult to transfer large genomic DNA molecules into mammalian cells by this method. We might first digest the normal human genomic DNA by restriction enzymes, and then transfer the normal genomic DNA fragments into a patient's stem cells and somatic cells by electroporation in vitro. After a few days of in vitro cell culture in serum free media, the cells can be transplanted into the same patient. Of course, before conducting human clinical

Whole genomic DNA molecules are too big to be transfected into cells directly. Normal genomic DNA can be digested by a few restriction enzymes first, and then the purified genomic DNA fragments can be transfected into stem cells and somatic cells from patients.

deleted from the 116 kb gene cluster by microinjection (J. Gao et al., 2005).

cells are sufficient to improve a patient's physiological function significantly.

trials, this kind of experiment should be performed in animal models first.

**3.4 Microinjection** 

et al., 1997).

al., 1983).

**3.5 Electroporation** 

**3.6 Genomic DNA Transfection**

Molecules commonly used for transfection are smaller than 10 kb; transfection efficiency is very low with plasmids of 12 kb or bigger (Campeau et al., 2001; Cartier & Reszka, 2002). Transfection is a relatively simple, easy, and convenient method to transfer a foreign DNA into a cell, but the current transfection methods cannot satisfy our needs when we want to transfer large DNA fragments. We have to improve the transfection efficiency, and new methods and advanced techniques are needed to transfer large genomic DNA fragments. A cell culture medium with a little bit lower osmotic pressure can cause cell osmotic swelling, and the cells become bigger, cell membrane permeability is increased, the nuclear pores might become bigger also. Therefore, bigger size of DNA molecules might be easier to enter the swelling cells and reach the genomic DNA inside the nucleus. After transfection, the transfected cells are grown in a cell culture medium with normal osmotic pressure for a period of time, and let the cells to recover to normal. The recovered transfected cells can be transplanted into animal models of a genetic disease.
