**3.3 Cell membrane and nuclear envelope**

Both a eukaryotic cell and a prokaryotic cell have a flexible lipid bilayer plasma membrane that controls movement of molecules in and out of the cell. A eukaryotic cell has a nucleus, while a prokaryotic cell does not have a nucleus; this is the characteristic difference between a eukaryotic cell and a prokaryotic cell.

The eukaryotic cell nucleus is surrounded by a nuclear envelope with nuclear pores. The nuclear envelope has two layers: the out nuclear membrane which faces the cytoplasm, and the inner nucleic membrane which faces the nucleoplasm. The nuclear pores are formed by nuclear pore complexes (NPCs) that span the double lipid bilayer of the nuclear envelope. The NPCs are formed by about 30 proteins. NPCs are gatekeepers of the nucleus (Alber et al., 2007; D'Angelo et al., 2006; D'Angelo & Hetzer, 2008; Devos et al., 2006; Fernandez-Martinez & Rout, 2009; Lam & Dean, 2010; Terry et al., 2007; Theerthagiri et al., 2010; E. Tran & Wente, 2006).

Ions and small molecules and DNA smaller than 200 bp can diffuse through the nuclear pore freely; while the transport of DNA molecules between 310 bp and 1500 bp from the cytosol to the nucleus is through an active transport process. DNA greater than 2 kb can rarely be seen in the nucleus (Cartier & Reszka, 2002; Hagstrom et al., 1997; Ludtke et al., 1999).

A foreign DNA molecule has to go through the human cell membrane, cytoplasm, and the nuclear envelope to reach the genomic DNA in the nucleus. This process can be performed and prompted by microinjection, electroporation, and transfection.

Gene Therapy of Some Genetic Diseases by Transferring

transplanted into animal models of a genetic disease.

chromosomes.

heterozygotes.

Akhunov, 2005; S. Huang et al., 2002).

**3.7 Genome sequencing and human genetic variation** 

Normal Human Genomic DNA into Somatic Cells and Stem Cells from Patients 113

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

Human somatic cells are diploid, each somatic cell has 23 homologous chromosome pairs (46 chromosomes), 23 of the chromosomes are from a sperm of the father and other 23 chromosomes are from an egg of the mother. The paired homologous chromosomes are similar in length, except the pair of X and Y chromosomes, an X chromosome is much longer than a Y chromosome. The human sperm cells and egg cells are haploid-each of them has 23

Different species of animals or plants have different number of chromosomes. A chimpanzee has 48 chromosomes (Young et al., 1960), a dog has 78, a chicken has 78, a pig has 38, a cat has 38, a horse has 64, a cow has 60, a goat has 60, a sheep has 54, a mouse has 40, and a rat has 42 chromosomes separately (O'Brien et al., 1999); and a silkworm has 28 chromosomes (International Silkworm Genome Consortium, 2008; Xia et al., 2004). Wheat has three ploidy levels: diploid wheat (Triticum urartu, Aegilops speltoides, and Ae. tauschii) has 14 (2x), tetraploid wheat (Triticum turgidum ssp. dicoccoides) has 28 (4x), and hexaploid wheat (Triticum aestivum) has 42 (6x) chromosomes respectively; diploid wheat is the ancestor of the tetraploid and hexaploid wheat (Akhunov et al., 2005, 2010; Dvorak &

Each human gene has two alleles, one allele on each chromosome of the homologous pair. If the 2 alleles have the same sequence, they are called homozygotes; otherwise, they are called

A specific phenotype (trait) might be determined by two alleles (recessive) or by one allele (dominant). In autosomal recessive genetic diseases like cystic fibrosis, sickle-cell anemia, and fanconi anemia (except FANCB), if a mutant gene appears on both of the paired homologous chromosomes of a person, this person has the genetic disease; if the mutant gene occurs on one of the homologous chromosomes of a person, this person is a carrier of the genetic disease. In X-linked recessive genetic diseases like Fanconi anemia subtype B, Duchenne muscular dystrophy, and Wiskott-Aldrich syndrome, if a male's X chromosome carries the mutated gene, this male has the genetic disease; if one of a female's X chromosomes carries the mutated gene, this female is a carrier; if both of a female's X chromosomes carry the mutated gene, this female has the genetic disease. On the other hand, in autosomal dominant genetic diseases like Huntington's disease, it only needs one mutated allele on any of the two homologous chromosomes to have the related genetic

#### **3.4 Microinjection**

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 et al., 1997).

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 al., 1983).

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

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