Gene Targeted Knockout Mice Assess the Contribution of a Particular Gene

One of the limitations with transgenic mice is that the transgene is integrated randomly within the genome. This means that some transgenes insert in regions of DNA that are not transcriptionally active, and hence the gene is not expressed. To circumvent this limitation, researchers have developed a technique in which a desired gene is targeted to specific sites within the germ line of a mouse. The primary use of this technique has been to replace a normal gene with a mutant allele or a disrupted form of the gene, thus knocking out the gene's function. Transgenic mice that carry such a disrupted gene, called knockout mice, have been extremely helpful to immunologists trying to understand how the removal of a particular gene product affects the immune system. Various knockout mice are being used in immunologic research, including mice that lack particular cytokines or MHC molecules.

Production of gene-targeted knockout mice involves the following steps:

■ Isolation and culturing of embryonic stem (ES) cells from the inner cell mass of a mouse blastocyst

Mouse Pronuclear Embryo

Inject cloned DNA into one of the pronuclei

Implant injected eggs into oviduct of pseudo-pregnant female

Pseudo-pregnant female

Inject cloned DNA into one of the pronuclei

Implant injected eggs into oviduct of pseudo-pregnant female

Pseudo-pregnant female

Offspring

Offspring

About 10-30% of offspring contain transgene

About 10-30% of offspring contain transgene

Test for presence of transgene

Test for presence of transgene

Breed transgenics

Breed transgenics

FIGURE 23-15

General procedure for producing transgenic mice. Fertilized eggs are collected from a pregnant female mouse. Cloned DNA (referred to as the transgene) is microinjected into one of the pronuclei of a fertilized egg. The eggs are then implanted into the oviduct of pseudopregnant foster mothers (obtained by mating normal females with a sterile male). The transgene will be incorporated into the chromosomal DNA of about 10%—30% of the offspring and will be expressed in all of their somatic cells. If a tissue-specific promoter is linked to a transgene, then tissue-specific expression of the transgene will result.

(a) Formation of recombinant ES cells

(a) Formation of recombinant ES cells

Recombinant Genes

Homologous recombination

Nonhomologous recombination

Homologous recombination

ES cell 'DNA"

Gene-targeted insertion

Nonhomologous recombination

Random insertion neoR tkHVS

(b) Selection of ES cell carrying knockout gene

Nonrecombinant cells Recombinants

Recombinants with with random. S (S (Ö) ^ ® gene-targeted

Treat with neomycin (nonrecombinant ES cells die)

Treat with gancyclovir (nonhomologous ES recombinant cells die)

CS S

Homologous ES recombinants with targeted disruption in gene X survive

FIGURE 23-16

Formation and selection of mouse recombinant ES

cells in which a particular target gene is disrupted. (a) In the engineered insertion construct, the target gene is disrupted with the neoR gene, and in only about 1% of the cells, with nonhomologous recombination much more frequent than homologous recombination. (b) Selection with the neomycin-like drug G418 will kill any nonrecombinant ES cells the thymidine kinase tk gene is located outside the target gene. The because they lack the neo gene. Selection with gancyclovir will kill the construct is transfected into cultured ES cells. If homologous recombination occurs, only the target gene and the neoR gene will be inserted into the chromosomal DNA of the ES cells. If nonhomologous recombination occurs, all three genes will be inserted. Recombination occurs nonhomologous recombinants carrying the tk gene, which confers sensitivity to gancyclovir. Only the homologous ES recombinants will survive this selection scheme. [Adapted from H. Lodish et al, 1995, Molecular Cell Biology, 3rd ed., Scientific American Books.]

■ Introduction of a mutant or disrupted gene into the cultured ES cells and selection of homologous recombinant cells in which the gene of interest has been knocked out (i.e., replaced by a nonfunctional form of the gene)

■ Injection of homologous recombinant ES cells into a recipient mouse blastocyst and surgical implantation of the blastocyst into a pseudo-pregnant mouse

■ Mating of chimeric offspring heterozygous for the disrupted gene to produce homozygous knockout mice

The ES cells used in this procedure are obtained by culturing the inner cell mass of a mouse blastocyst on a feeder layer of fibroblasts or in the presence of leukemia-inhibitory factor. Under these conditions, the stem cells grow but remain pluri-potent and capable of later differentiating in a variety of directions, generating distinct cellular lineages (e.g., germ cells, myocardium, blood vessels, myoblasts, nerve cells). One of the advantages of ES cells is the ease with which they can be genetically manipulated. Cloned DNA containing a desired gene can be introduced into ES cells in culture by various transfection techniques. The introduced DNA will be inserted by recombination into the chromosomal DNA of a small number of ES cells.

The insertion constructs introduced into ES cells contain three genes: the target gene of interest and two selection genes, such as neoR, which confers neomycin resistance, and the thymidine kinase gene from herpes simplex virus (tAHSV), which confers sensitivity to gancyclovir, a cytotoxic nucleotide analog (Figure 23-16a). The construct often is engineered with the target-gene sequence disrupted by the neoR gene and with the tkHSV gene at one end, beyond the sequence of the target gene. Most constructs will insert at random by nonhomolo-gous recombination rather than by gene-targeted insertion through homologous recombination. As illustrated in Figure 23-16b, a two-step selection scheme is used to obtain those ES cells that have undergone homologous recombination, whereby the disrupted gene replaces the target gene.

The ES cells obtained by this procedure are heterozygous for the knockout mutation in the target gene. These cells are clonally expanded in cell culture and then injected into a

Inject ES cells into blastocoel cavity of early embryo. ES cells are heterozygous for knockout mutation in gene X and homozygous for black coat color; embryo is homozygous for white coat color

Embrio Tranfer Mouse
Surgically transfer embryo into pseudopregnant mouse
Knockout Mice Procedure

Chimeric progeny have black-and-white coats. White areas are derived from recipient blastocoel cells, black areas from ES cells

Mate chimeric mice to homozygous white mice

Chimeric progeny have black-and-white coats. White areas are derived from recipient blastocoel cells, black areas from ES cells

Mate chimeric mice to homozygous white mice

FIGURE 23-17

General procedure for producing homozygous knockout mice. ES cells homozygous for a marker gene (e.g., black coat color) and heterozygous for a disrupted target gene (see Figure 23-18) are injected into an early embryo homozygous for an alternate marker (e.g., white coat color). The chimeric transgenic offspring, which have black-and-white coats, then are mated with homozygous white mice. The all-black progeny from this mating have ES-derived cells in their germ line, which are heterozygous for the disrupted target gene. Mating of these mice with each other produces animals homozygous for the disrupted target gene, that is, knockout mice. [Adapted from M. R. Capecchi, 1989, Trends Genet. 5:70.]

Black progeny develop from germ-line cells derived from ES cells and are heterozygous for disrupted gene X

mouse blastocyst, which subsequently is implanted into a pseudo-pregnant female. The transgenic offspring that develop are chimeric, composed of cells derived from the genetically altered ES cells and cells derived from normal cells of the host blastocyst. When the germ-line cells are derived from the genetically altered ES cells, the genetic alteration can be passed on to the offspring. If the recombinant ES cells are homozy-gous for black coat color (or other visible marker) and they are injected into a blastocyst homozygous for white coat color, then the chimeric progeny that carry the heterozygous knockout mutation in their germ line can be easily identified (Figure 23-17). When these are mated with each other, some of the offspring will be homozygous for the knockout mutation.

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