Biomedical Engineering Reference
In-Depth Information
mice [11, 12], while the second was the introduction of specific mutations into a chosen
gene by homologous recombination in the cultured mouse ES cells [13, 14]. The first
reports using homologous recombination in ES cells to generate gene-targeted mice followed
thereafter [15-18].
Gene targeting in mice relies on the unique attributes of mouse ES cells, which are
derived from the pluripotent, uncommitted cells of the inner cell mass of pre-implantation
blastocysts. They can be manipulated in culture, introduced into a pre-implantation blas-
tocyst embryo and allowed to mature in a foster mother. These pluripotent cells have the
ability to contribute efficiently to the formation of both somatic and germline tissues after
reintroduction into a blastocyst. This capacity is highly dependent on culture conditions that
keep ES cells in an uncommitted, undifferentiated state. Differentiation inhibitory signals
are provided by mouse embryonic fibroblast (MEF) feeder cells that also act as a matrix
for ES cell adherence and by addition of leukemia inhibitory factor (LIF) to the culture
medium [19]. While the R1 ES cells described in these protocols do rely on feeder cells,
several ES cell lines are available that do not have this requirement, and thus require less
labor-intensive cultivation.
Targeted mutations can be introduced in ES cells by homologous recombination of a
suitable targeting vector (see Figure 10.1). Homologous recombination in mammalian cells
occurs at a very low frequency; therefore, positive-negative selection was developed as a
method for enrichment of cells in which homologous targeting has occurred [20]. Incor-
poration of the targeting vector into the genome is positively selected for by addition of
the antibiotic G418/geneticin, due to the presence of a cassette carrying the neomycin
resistance gene under the control of a strong promoter. For negative selection, the herpes
simplex virus thymidine kinase (TK) gene is found at the end of the linearized targeting
construct. Cells undergoing homologous recombination will lose the TK gene, while those
with random insertion of the targeting vector are eliminated by applying the toxic nucle-
oside analog, FIAU [1-(2-deoxy-2-fluoro-
β
-D-arabino-furanosyl)-5-iodouracil]. Diphtheria
toxin A (DT-A) has also been successfully used as a negative selection marker [21].
The following factors have been shown to influence targeting frequency:
1 The recombination rate increases with total length of homology between a vector and its
target locus, up to about 10 kb [22]. Here, this is maximized by the use of a 6-10 kb long
homology arm (LHA) and an
1 kb short homology arm (SHA) flanking the neomycin
resistance cassette (Figure 10.1). The LHA can be cloned through retrieval of DNA
from a BAC, described in Protocols 10.1-10.4, while the SHA is cloned using standard
polymerase chain reaction (PCR)-based methods.
2 Targeting frequency increases when the homology regions of the gene of interest are
isolated from the same genetic background (isogenic) as the ES cells to be used [23].
R1 ES cells are derived from the 129 genetic background [24]; however, cell lines from
other mouse strains such as C57BL/6 and BALB/c are also available and may be used
[25, 26].
3 Targeting frequency is locus dependent; thus, while the methods described here maximize
the efficiency of homologous recombination, factors such as chromatin structure and
accessibility make the overall targeting frequency difficult to predict for a given gene.
∼
Several methods can be used to screen for ES cell clones with the desired recombination
event, including PCR and optimized mini-Southern blot. The strategy outlined here takes
