Cell culture has become one of the most powerful techniques, as cultured cells are being

used in a diversity of biologic felds ranging from biochemistry to molecular and cellular
biology.24 Through their ability to be maintained in vitro, cells can be manipulated by the
introduction of genes of interest (cell transfection) and be transferred into in vivo biologic
receivers (cell transplantation) to study the biologic efect of the interested genes (Fig. 15-
21). In general, cell culture procedures are simple and straightforward. In the laboratory,
cells are cultured either as a monolayer (in which cells grow as one layer on culture dishes)
or in suspension.
It is important to know the wealth of information concerning cell culturing before attempting
the procedure. For example, conditions of culture will depend on the cell types to be
cultured (e.g., origins of the cells such as epithelial or fbroblasts, or primary vs.
immortalized/transformed cells). It also is necessary to use special culture medium that
has been used to establish the cell line (if it is a cell line), including the type and
concentration of serum used to maintain the growth of cells in vitro. If primary cells are
derived from human patients or animals, some commercial resources have a variety of
culture media available for testing. Generally, cells are manipulated in a sterile hood and
the working surfaces are wiped with 80% ethyl alcohol solution. Cultured cells are
maintained in a humidifed carbon dioxide incubator at 37C (98.6F), and need to be
examined daily under an inverted microscope to check for possible contamination and
confuency (the area cells occupy on the dish). As a general rule, cells should be fed with
fresh medium every 2 to 3 days and split when they reach confuency. Depending upon the
growth rate of cells, the actual time and number of plates required to split cells in two
varies from cell line to cell line. Splitting a monolayer requires the detachment of cells from
plates by using a trypsin treatment, of which concentration and time period vary depending
on cell lines. If cultured cells grow continuously in suspension, they are split or subcultured
by dilution.
Because cell lines may change their properties when cultured, it is not possible to maintain
cell lines in culture indefnitely. Therefore, it is essential to store cells at various time
passages for future use. The common procedure is to use cryopreservation. The solution
for cryopreservation is fetal calf serum containing 10% dimethyl sulfoxide or glycerol,
stored in liquid nitrogen [196C (320.8F)]. Cells can be stored for many years using this
method.
CELL TRANSFECTION
Cells are cultured for two reasons: to maintain and to manipulate them (see Fig. 15-21).
The transfer of foreign macromolecules, such as nucleic acid, into living cells provides an
efcient method for studying a variety of cellular processes and functions at the molecular
level. DNA transfection has become an important tool for studying the regulation and
function of genes. The cDNA to be expressed should be in a plasmid vector, behind an
appropriate promoter working in mammalian cells (e.g., the constitutively active
cytomegalovirus promoter or inducible promoter). Depending on the cell type, many ways
of introducing DNA into mammalian cells have been developed. Commonly used
approaches include calcium phosphate, electroporation, liposome-mediated transfection,
the nonliposomal formulation, and the use of viral vectors. These methods have shown
variable success when attempting to transfect a wide variety of cells. Transfection can be
performed in the presence or absence of serum. It is suggested to test the transfection
efciency of cell lines of interest by comparing transfection with several diferent
approaches. For a detailed transfection protocol, it is best to follow the manufacturer's
instructions for the particular reagent. General considerations for a successful transfection
depend on several parameters, such as the quality and quantity of DNA and cell culture
(type of cell and growth phase). To minimize variations in both of these in transfection
experiments, it is best to use cells that are healthy, proliferate well, and are plated at a
constant density. After DNA is introduced into the cells, it is normally maintained
epitopically in cells and will be diluted while host cells undergo cell division. Therefore,
functional assays should be performed 24 to 72 hours after transfection, also termed
transient transfection. In many applications, it is important to study the long-term efects of
DNA in cells by stable transfection. Stable cell clones can be selected for DNA integration
into the host cell genome, when plasmids carry an antibiotic-resistant marker. In the
presence of antibiotics, only those cells that continuously carry the antibiotic-resistant
marker (after generations of cell division) can survive. One application of stable
transfection is the generation of transgenic or knockout mouse models, in which the
transgene has to be integrated in the mouse genome. Stable cells also can be
transplanted into host organs.
Genetic Manipulations
Understanding how genes control the growth and diferentiation of the mammalian
organism has been the most challenging topic of modern research. It is essential for us to
understand how genetic mutations and chemicals lead to the pathologic condition of
human bodies. The knowledge and ability to change the genetic program will inevitably
make a great impact on society and have far-reaching efects on how we think of
ourselves.
The mouse has become frmly established as the primary experimental model for studying
how genes control mammalian development. Genetically altered mice are powerful model
systems in which to study the function and regulation of genes.25 The gene function can
be studied by creating mutant mice through homologous recombination (gene knockout). A
gene of interest also can be introduced into the mouse (transgenic mouse) to study its
efect on development or diseases. As mouse models do not precisely represent human
biology, genetic manipulations of human somatic or ES cells provide a great means for the
understanding of the molecular networks in human cells. In all cases, the gene to be
manipulated must frst be cloned. Gene cloning has been made easy by recombinant DNA
technology and the availability of human and mouse genomes (see the Human Genome
section). The following section briefy describes the technologies and the principles behind
them.
TRANSGENIC MICE
During the past 20 years, DNA cloning and other techniques have allowed the introduction
of new genetic material into the mouse germline. As early as 1980, the frst genetic
material was successfully introduced into the mouse germline by using pronuclear
microinjection of DNA (Fig. 15- 22). These animals, called transgenic, contain foreign DNA
within their genomes. In simple terms, a transgenic mouse is created by the microinjection
of DNA into the one-celled mouse embryo, allowing the efcient introduction of cloned
genes into the developing mouse somatic tissues, as well as into the germline.
Designs of a Transgene
The transgenic technique has proven to be extremely important for basic investigations of
gene regulation, creation of animal models of human disease, and genetic engineering of
livestock. The design of a transgene construct is a simple task. Like constructs used in cell
transfection, a simple transgene construct consists of a protein-encoding gene and a
promoter that precedes it. The most common applications for the use of transgenic mice
are similar to those in the cell culture system: (a) to study the functions of proteins encoded
by the transgene and (b) to analyze the tissue-specifc and developmental-stagespecifc
activity of a gene promoter. Examples of the frst application include overexpression of
oncogenes, growth factors, hormones, and other key regulatory genes, as well as genes of
viral origins. Overexpression of the transgene normally represents gain-of-function
mutations. The tissue distribution or expression of a transgene is determined primarily by
cis- acting promoter enhancer elements within or in the immediate vicinity of the genes
themselves. Thus, controlled expression of the transgene can be made possible by using
an inducible or tissue-specifc promoter. Furthermore, transgenic mice carrying dominant
negative mutations of a regulatory gene also have been generated. For example, a
truncated growth factor receptor that can bind to the ligand, but loses its catalytic activity
when expressed in mice, can block the growth factor binding to the endogenous protein. In
this way, the transgenic mice exhibit a loss of function of phenotype, possibly resembling
the knockout of the endogenous gene. The second application of the transgenic
expression is to analyze the gene promoter of interest. The gene promoter of interest
normally is fused to a reporter gene that encodes -galactosidase (also called LacZ),
luciferase, or green fuorescence protein. Chemical staining of LacZ activity or detection of
chemiluminescence/fuorescence can easily visualize the expression of the reporter gene.
The amount of the reporter gene activity represents the activity of the promoter, and thus,
reporter activities are tightly correlated to expression of the gene in which the promoter is
used to drive the reporter gene expression.
Production of Transgenic Mice
The success of generating transgenic mice is largely dependent upon the proper quality
and concentration of the DNA supplied for microinjection. For DNA to be microinjected into
mouse embryos, it should be linearized by restriction digestion to increase the chance of
proper transgene integration. Concentration of DNA should be accurately determined. Mice
that develop from injected eggs often are termed founder mice.
Genotyping of Transgenic Mice
The screening of founder mice and the transgenic lines derived from the founders is
accomplished by determining the integration of the injected gene into the genome. This
normally is achieved by performing PCR or Southern blot analysis with a small amount of
DNA extracted from the mouse tail. Once a given founder mouse is identifed to be
transgenic, it will be mated to begin establishing a transgenic line.
Analysis of Phenotype of Transgenic Mice
Phenotypes of transgenic mice are dictated by both the expression pattern and biologic
functions of the transgene. Depending on the promoter and the transgene, phenotypes can
be predictable or unpredictable. Elucidation of the functions of the transgene-encoded
protein in vitro often ofers some clue to what the protein might do in vivo. When a
constitutively active promoter is used to drive the expression of transgenes, mice should
express the gene in every tissue; however, this mouse model may not allow the
identifcation and study of the earliest events in disease pathogenesis. Ideally, the use of
tissue-specifc or inducible promoter allows one to determine if the pathogenic protein
leads to a reversible or irreversible disease process. For example, rat insulin promoter can
target transgene expression exclusively in the -cells of pancreatic islets. The phenotype of
insulin promoter-mediated transgenic mice is projected to afect the function of human
-cells.