Principles
The diagnosis of a genetic disease requires a
systematic approach that takes clinical and
genetic considerations into account. Whereas
clinical medicine tends to classify diseases according
to organ system, age of onset, gender,
or primary method of detection (radiology,
imaging techniques), medical genetics, like
pathology, is oriented towards the basic cause
or lesion, in this case the gene or genes affected
by a relevant genetic change. Genetic diagnosis
is based on an interdisciplinary analysis of all
clinical and laboratory data from a genetic perspective.
Sunday, April 12, 2009
Genetic diagnosis, a multistep procedure
The phenotype, which is the clinical manifestations
including individual and family history in
medical terms, is the starting point. The first
decision the medical geneticist must make is
whether a pattern of manifestations can be recognized.
Tools that can assist in this decision include
training and personal experience; appropriate
textbooks and other literature; and online
search systems such as OMIM, MEDLINE,
PubMed, POSSUM, London Dysmorphology
Data Base for congenital malformations, and cytogenetic
databases.
including individual and family history in
medical terms, is the starting point. The first
decision the medical geneticist must make is
whether a pattern of manifestations can be recognized.
Tools that can assist in this decision include
training and personal experience; appropriate
textbooks and other literature; and online
search systems such as OMIM, MEDLINE,
PubMed, POSSUM, London Dysmorphology
Data Base for congenital malformations, and cytogenetic
databases.
disease pattern can be recognized
If a disease pattern can be recognized, the next
decision concerns the category of disease. Although
difficult to establish in practice, the disease
category is important for the next steps to
be taken. For this purpose, the McKusick catalog
of human genes and diseases, Mendelian Inheritance
in Man (MIM) and its online version
(OMIM) are indispensable. The possibility of
genetic heterogeneity must be considered at
this stage. The term genetic heterogeneity refers
to a phenotype (disease) that has different
causes. A particular phenotype may be caused
by mutations at different loci (locus heterogeneity)
or by different mutant alleles at the same
locus (allele heterogeneity). All genetic diagnostic
procedures should be preceded by genetic
counseling, which properly includes obtaining
the (informed) consent of the persons involved.
decision concerns the category of disease. Although
difficult to establish in practice, the disease
category is important for the next steps to
be taken. For this purpose, the McKusick catalog
of human genes and diseases, Mendelian Inheritance
in Man (MIM) and its online version
(OMIM) are indispensable. The possibility of
genetic heterogeneity must be considered at
this stage. The term genetic heterogeneity refers
to a phenotype (disease) that has different
causes. A particular phenotype may be caused
by mutations at different loci (locus heterogeneity)
or by different mutant alleles at the same
locus (allele heterogeneity). All genetic diagnostic
procedures should be preceded by genetic
counseling, which properly includes obtaining
the (informed) consent of the persons involved.
Protein truncation test (PTT)
This is a test for frameshift, splice, or nonsense
mutations that leads to a truncated protein due
to an early stop codon created downstream of
the mutation. The truncated protein is detected
in an assay based on an in-vitro translation system.
The translation will be interrupted at a
premature stop codon resulting from the mutation.
The size of the newly translated protein is
determined by gel electrophoresis. PTT detects
the approximate location of the mutation as reflected
by the size of the mutant protein. PTT is
useful in studying genes with frequent nonsense
mutations, such as the APC, BRCA1, and
BRCA2 genes. However, it cannot be applied for
genes with frequent missense mutations.
mutations that leads to a truncated protein due
to an early stop codon created downstream of
the mutation. The truncated protein is detected
in an assay based on an in-vitro translation system.
The translation will be interrupted at a
premature stop codon resulting from the mutation.
The size of the newly translated protein is
determined by gel electrophoresis. PTT detects
the approximate location of the mutation as reflected
by the size of the mutant protein. PTT is
useful in studying genes with frequent nonsense
mutations, such as the APC, BRCA1, and
BRCA2 genes. However, it cannot be applied for
genes with frequent missense mutations.
Detection of Mutations without Sequencing
In addition to the detection of mutations by
different DNA fragments in Southern blots
(p. 62), there are methods based on differences
in the hybridization of mutated and normal segments
of DNA. Incomplete hybridization is
determined by using short segments of singlestranded
DNA (oligonucleotides) with a
sequence complementary to the investigated
region (see A). Other methods are based on
demonstrating incomplete hybridization with
mRNA (see B) or on the fact that a hybridized
segment of normal and mutant DNA is less
stable than normal DNA.
different DNA fragments in Southern blots
(p. 62), there are methods based on differences
in the hybridization of mutated and normal segments
of DNA. Incomplete hybridization is
determined by using short segments of singlestranded
DNA (oligonucleotides) with a
sequence complementary to the investigated
region (see A). Other methods are based on
demonstrating incomplete hybridization with
mRNA (see B) or on the fact that a hybridized
segment of normal and mutant DNA is less
stable than normal DNA.
Detection of a point mutation by oligonucleotides
Short segments of DNA (oligonucleotides) are
used to determine whether there is a mutation
in a segment of DNA (1, normal DNA; 2, mutation
from G to A). An oligonucleotide is a synthetically
produced DNA segment about 20 nucleotides
long; its sequence is complementary
to a corresponding segment of the investigated
gene. It hybridizes completely with its complementary
segment (3). If a mutation, here
from G to A (1), is located in this region, hybridization
will not be perfect at this site (mismatch)
(4). On the other hand, an oligonucleotide
that is complementary to the DNA segment
with the mutation will hybridize
completely (allele-specific oligonucleotide,
ASO) (5). This hybridizes incompletely with the
normal DNA (6). By parallel use of both nucleotides,
mutant and nonmutant DNA can be
differentiated. The test results (7) show the hybridization
of mutated DNA and of control DNA
with the allele-specific oligonucleotides (ASO 1
for the control, ASO 2 for the mutation).
used to determine whether there is a mutation
in a segment of DNA (1, normal DNA; 2, mutation
from G to A). An oligonucleotide is a synthetically
produced DNA segment about 20 nucleotides
long; its sequence is complementary
to a corresponding segment of the investigated
gene. It hybridizes completely with its complementary
segment (3). If a mutation, here
from G to A (1), is located in this region, hybridization
will not be perfect at this site (mismatch)
(4). On the other hand, an oligonucleotide
that is complementary to the DNA segment
with the mutation will hybridize
completely (allele-specific oligonucleotide,
ASO) (5). This hybridizes incompletely with the
normal DNA (6). By parallel use of both nucleotides,
mutant and nonmutant DNA can be
differentiated. The test results (7) show the hybridization
of mutated DNA and of control DNA
with the allele-specific oligonucleotides (ASO 1
for the control, ASO 2 for the mutation).
Demonstration of a point mutation
Demonstration of a point mutation
by ribonuclease A cleavage
The basis for this method is that a normal DNA
strand hybridizes completely with mRNA from
that region. Completely hybridized DNA and
mRNA are protected from the effects of the
RNA-splitting enzyme ribonuclease A (ribonuclease
protection assay). Hybridization is incomplete
in the area of a mutation. In this region,
mRNA will be cleaved by ribonuclease A
(RNAase A). This can be demonstrated by
Southern blot. There will be two fragments
formed that together correspond to the size of
the completely hybridized fragment
by ribonuclease A cleavage
The basis for this method is that a normal DNA
strand hybridizes completely with mRNA from
that region. Completely hybridized DNA and
mRNA are protected from the effects of the
RNA-splitting enzyme ribonuclease A (ribonuclease
protection assay). Hybridization is incomplete
in the area of a mutation. In this region,
mRNA will be cleaved by ribonuclease A
(RNAase A). This can be demonstrated by
Southern blot. There will be two fragments
formed that together correspond to the size of
the completely hybridized fragment
Denaturing gradient gel electrophoresis
This method exploits differences in the stability
of DNA segments with and without mutation.
While double-stranded DNA of a control person
is completely complementary (homoduplex), a
mutation leads to a mismatch at the site of mutation
(heteroduplex). This DNA is less stable
than completely complementary DNA strands
(it has a lower melting point). If normal DNA
(control) and DNAwith the mutation are placed
in a gel with an increasing concentration
gradient of formamide (denaturing gradient
gel), the mutant and normal DNA can subsequently
be differentiated in a Southern blot.
The normal DNA remains stable to higher concentrations
of formamide and migrates farther
than mutant DNA, which dissociates earlier and
therefore does not migrate as far.
of DNA segments with and without mutation.
While double-stranded DNA of a control person
is completely complementary (homoduplex), a
mutation leads to a mismatch at the site of mutation
(heteroduplex). This DNA is less stable
than completely complementary DNA strands
(it has a lower melting point). If normal DNA
(control) and DNAwith the mutation are placed
in a gel with an increasing concentration
gradient of formamide (denaturing gradient
gel), the mutant and normal DNA can subsequently
be differentiated in a Southern blot.
The normal DNA remains stable to higher concentrations
of formamide and migrates farther
than mutant DNA, which dissociates earlier and
therefore does not migrate as far.
Chromosomal Location of Monogenic Diseases
Chromosomal Location of
Human Genetic Diseases
Nowhere is the growth of knowledge about disease-
causing mutations in human genes more
apparent than in the mapping of their locations
to specific sites on individual chromosomes.
This progress is documented in twelve published
editions of Mendelian Inheritance in
Man. A Catalog of Human Genes and Disorders
(MIM) by Victor A. McKusick, M.D., at the Johns
Hopkins University School of Medicine. Initiated
in the early 1960s, its first edition in 1966
contained a total of 1487 entries without a
single autosomal gene mapped. This was
achieved in 1968 at the time of the second edition
(1545 entries). The subsequent editions reveal
an entry-doubling time of about 15 years
(3368 in the 6th edition (1983), 5710 entries in
the 10th edition (1992), 8587 entries in the 12th
edition (1998) and 10848 on 21 September
1999 (Hamosh et al., 2000). Since 1987 the
McKusick catalog is internationally available
online from the National Library of Medicine
Human Genetic Diseases
Nowhere is the growth of knowledge about disease-
causing mutations in human genes more
apparent than in the mapping of their locations
to specific sites on individual chromosomes.
This progress is documented in twelve published
editions of Mendelian Inheritance in
Man. A Catalog of Human Genes and Disorders
(MIM) by Victor A. McKusick, M.D., at the Johns
Hopkins University School of Medicine. Initiated
in the early 1960s, its first edition in 1966
contained a total of 1487 entries without a
single autosomal gene mapped. This was
achieved in 1968 at the time of the second edition
(1545 entries). The subsequent editions reveal
an entry-doubling time of about 15 years
(3368 in the 6th edition (1983), 5710 entries in
the 10th edition (1992), 8587 entries in the 12th
edition (1998) and 10848 on 21 September
1999 (Hamosh et al., 2000). Since 1987 the
McKusick catalog is internationally available
online from the National Library of Medicine
OMIM
Regularly updated, OMIM has become a major
source of information on human genes and
genetic diseases. Each entry has a unique 6-
digit identifying number and is assigned to one
of five catalogs according to genetic category:
(1) autosomal dominant, (2) autosomal recessive,
(3) X-chromosomal, (4) Y-chromosomal,
and (5) mitochondrial. Autosomal entries initiated
since 1994 begin with the digit 6. The
McKusick catalog has provided a systematic
basis for the genetics of man comparable to the
first periodic table of chemical elements by
Dimitrij I. Mendelyev in 1869 or to the
“Chronologisch-thematisches Verzeichnis
sämtlicher Tonwerke Wolfgang Amade
Mozarts” by Ludwig Alois Ferdinand Köchel in
1862.
source of information on human genes and
genetic diseases. Each entry has a unique 6-
digit identifying number and is assigned to one
of five catalogs according to genetic category:
(1) autosomal dominant, (2) autosomal recessive,
(3) X-chromosomal, (4) Y-chromosomal,
and (5) mitochondrial. Autosomal entries initiated
since 1994 begin with the digit 6. The
McKusick catalog has provided a systematic
basis for the genetics of man comparable to the
first periodic table of chemical elements by
Dimitrij I. Mendelyev in 1869 or to the
“Chronologisch-thematisches Verzeichnis
sämtlicher Tonwerke Wolfgang Amade
Mozarts” by Ludwig Alois Ferdinand Köchel in
1862.
Saturday, April 11, 2009
Introduction
Disorders affecting craniofacial development represent a large fraction of birth
defects (1). The advent of transgenic and gene knockout technology has led to the
identification of a variety of genes that have roles in craniofacial development.
These technologies, together with the tools of human genetics, are being used to recreate
specific human genetic defects in the mouse, providing an understanding of the
pathophysiology of craniofacial disorders and laying the foundation for improvements
in therapies.
defects (1). The advent of transgenic and gene knockout technology has led to the
identification of a variety of genes that have roles in craniofacial development.
These technologies, together with the tools of human genetics, are being used to recreate
specific human genetic defects in the mouse, providing an understanding of the
pathophysiology of craniofacial disorders and laying the foundation for improvements
in therapies.
Preliminary Considerations
Nature of Genetic Defect
The nature of the human genetic defect informs the approach to modeling the disorder.
If the defect is caused by a recessive loss of function mutation or a dominant
hapoloinsufficiency, then the modeling of the disorder will require inactivating or
attenuating the function of the gene in question. If the defect is caused by a dominant
gain of function mutation, then a transgenic approach may provide a suitable model.
Subsumed in the gain of function mechanism are dominant activating mutations, which
enhance the function of the gene product, as well as dominant negative mutations,
which inactivate the protein in question and also abrogate the activity of the wild-type
protein. Both mechanisms can be modeled by transgenic approaches. In the case of the
dominant activating mutation, it may not be necessary to express the mutant form of
the protein; simple overexpression of the wild-type gene may be sufficient to produce
a phenotype. In the case of a dominant negative mechanism, expression of the mutant
protein would be necessary to produce the phenotype.
The nature of the human genetic defect informs the approach to modeling the disorder.
If the defect is caused by a recessive loss of function mutation or a dominant
hapoloinsufficiency, then the modeling of the disorder will require inactivating or
attenuating the function of the gene in question. If the defect is caused by a dominant
gain of function mutation, then a transgenic approach may provide a suitable model.
Subsumed in the gain of function mechanism are dominant activating mutations, which
enhance the function of the gene product, as well as dominant negative mutations,
which inactivate the protein in question and also abrogate the activity of the wild-type
protein. Both mechanisms can be modeled by transgenic approaches. In the case of the
dominant activating mutation, it may not be necessary to express the mutant form of
the protein; simple overexpression of the wild-type gene may be sufficient to produce
a phenotype. In the case of a dominant negative mechanism, expression of the mutant
protein would be necessary to produce the phenotype.
Design of Transgene
Assuming that a transgenic approach is likely to provide a model of the disorder
in question, the next step is to design a suitable vector for the expression of the
gene. A key element of the expression vector is the promoter. A few promoters that
direct expression to craniofacial tissues have been characterized in sufficient detail to
be useful. Table 1 describes these promoters and their expression patterns.
In general, the use of a promoter that mimics the expression of the endogenous gene
is ideal. This allows for the maximum precision in the modeling of the disorder. However,
a more general promoter can also be informative. As has been clearly demonstrated
from work in Drosophila and vertebrates, the ectopic expression of a gene can
show whether the gene has a dominant effect on cell fate or morphogenetic processes.
This information may prove helpful in the analysis of the pathophysiological mechanism.
For example, in Boston craniosynostosis, we used the CMV promoter to gener-ally overexpress Msx2 (9) and the Msx2 promoter to overexpress Msx2 at the sites of its
normal expression (9a). We showed that in general, CMV-driven overexpression
resulted in a craniosynostosis-like phenotype as well as ectopic cranial bone. Specific
overexpression under the control of Msx2 caused only the craniosynostosis-like condition.
These data suggested that Msx2 is sufficient to induce cells to an osteogenic fate
and thus provided a hypothesis as to how a dominant active mutation in Msx2 could
lead to craniosynostosis.
in question, the next step is to design a suitable vector for the expression of the
gene. A key element of the expression vector is the promoter. A few promoters that
direct expression to craniofacial tissues have been characterized in sufficient detail to
be useful. Table 1 describes these promoters and their expression patterns.
In general, the use of a promoter that mimics the expression of the endogenous gene
is ideal. This allows for the maximum precision in the modeling of the disorder. However,
a more general promoter can also be informative. As has been clearly demonstrated
from work in Drosophila and vertebrates, the ectopic expression of a gene can
show whether the gene has a dominant effect on cell fate or morphogenetic processes.
This information may prove helpful in the analysis of the pathophysiological mechanism.
For example, in Boston craniosynostosis, we used the CMV promoter to gener-ally overexpress Msx2 (9) and the Msx2 promoter to overexpress Msx2 at the sites of its
normal expression (9a). We showed that in general, CMV-driven overexpression
resulted in a craniosynostosis-like phenotype as well as ectopic cranial bone. Specific
overexpression under the control of Msx2 caused only the craniosynostosis-like condition.
These data suggested that Msx2 is sufficient to induce cells to an osteogenic fate
and thus provided a hypothesis as to how a dominant active mutation in Msx2 could
lead to craniosynostosis.
Msx2 promoter
The use of the Msx2 promoter was made possible by first mapping the promoter in
transgenic mice. This entailed fusing the promoter with a lacZ reporter and using such
constructs to produce transgenic mice. Although this can be labor intensive and expensive,
it is a necessary first step in identifying a promoter that can be used to overexpress
a putative disease gene.
transgenic mice. This entailed fusing the promoter with a lacZ reporter and using such
constructs to produce transgenic mice. Although this can be labor intensive and expensive,
it is a necessary first step in identifying a promoter that can be used to overexpress
a putative disease gene.
Production and Evaluation of Mice
We use standard methods the implantation of injected eggs into pseudopregnant females, and the
genotyping of resulting pups.
genotyping of resulting pups.
Choice of Stable vs Transient Transgenics
Transgenic mice are typically made as stable lines, which entails breeding founder
mice and evaluating the phenotype in the F1 or F2 generation. Alternatively, F0s may be
analyzed directly. Such a “transient” transgenic approach offers the advantage of relatively
quick results. A disadvantage is that the transgenic line is not maintained; therefore
further analysis of any phenotype will require more injections.
mice and evaluating the phenotype in the F1 or F2 generation. Alternatively, F0s may be
analyzed directly. Such a “transient” transgenic approach offers the advantage of relatively
quick results. A disadvantage is that the transgenic line is not maintained; therefore
further analysis of any phenotype will require more injections.
Evaluation of Phenotypes
After the transgene status of founder animals has been demonstrated and a breeding
strategy established, the next major issue facing an investigator is to identify any phenotypic
change in transgenic animals compared with their nontransgenic counterparts.
We outline several approaches, macroscopic and microscopic, that we have found useful
in pursuit of a complete characterization of the transgenic phenotype.
strategy established, the next major issue facing an investigator is to identify any phenotypic
change in transgenic animals compared with their nontransgenic counterparts.
We outline several approaches, macroscopic and microscopic, that we have found useful
in pursuit of a complete characterization of the transgenic phenotype.
LETHALITY
The first issue is whether overexpression of the transgene is lethal. This problem is
most acute when the transgene is generally overexpressed throughout development.
Lethality may be embryonic or postnatal. Demonstrating embryonic lethality entails
first genotyping progeny of a transgenic mating. The absence of transgenic mice after
screening a significant number of pups strongly suggests embryonic lethality. There
may also be a correlation between transgene dosage and lethality: homozygosity for
the transgene may be a lethal condition, whereas heterozygosity is not. If lethality is
encountered, sometimes simply screening a sufficiently large number of transgenics
will identify viable animals. Presumably such animals are viable because they express the
transgene at lower levels. Another approach is to try a different mouse strain. Outbred
and hybrid strains often reduce the severity of a transgenic phenotype.
most acute when the transgene is generally overexpressed throughout development.
Lethality may be embryonic or postnatal. Demonstrating embryonic lethality entails
first genotyping progeny of a transgenic mating. The absence of transgenic mice after
screening a significant number of pups strongly suggests embryonic lethality. There
may also be a correlation between transgene dosage and lethality: homozygosity for
the transgene may be a lethal condition, whereas heterozygosity is not. If lethality is
encountered, sometimes simply screening a sufficiently large number of transgenics
will identify viable animals. Presumably such animals are viable because they express the
transgene at lower levels. Another approach is to try a different mouse strain. Outbred
and hybrid strains often reduce the severity of a transgenic phenotype.
GROSS ANALYSIS OF CRANIOFACIAL PHENOTYPES
As a first step in the evaluation of morphological phenotypes, we generally carry out
alizarin red S and Alcian blue stains of whole skulls (11). Nontransgenic littermates are
used as controls. This technique can be easily used in conjunction with animal necropsy
to identify changes in the whole animal. For example, we knew that Msx2 was
expressed in abundant amounts in a wide variety of tissues outside the skull. Consequently,
overexpressing the Msx2 protein under the control of either the CMV promoter
or the Msx promoter might alter the developmental fate of noncalvarial tissues in
transgenic animals. To identify such changes, we undertook complete necropsies on
animals from each transgenic line from several selected stages of development. These
necropsy studies of visceral organs could be easily combined with other approaches,
such as preserving the bodies for alizarin red staining of the skeleton. In this manner,
alterations in the visceral organs or the skeletal body plan could be identified and an
approach developed to explore the role of the expression of the transgene in the development
of the affected tissue.
alizarin red S and Alcian blue stains of whole skulls (11). Nontransgenic littermates are
used as controls. This technique can be easily used in conjunction with animal necropsy
to identify changes in the whole animal. For example, we knew that Msx2 was
expressed in abundant amounts in a wide variety of tissues outside the skull. Consequently,
overexpressing the Msx2 protein under the control of either the CMV promoter
or the Msx promoter might alter the developmental fate of noncalvarial tissues in
transgenic animals. To identify such changes, we undertook complete necropsies on
animals from each transgenic line from several selected stages of development. These
necropsy studies of visceral organs could be easily combined with other approaches,
such as preserving the bodies for alizarin red staining of the skeleton. In this manner,
alterations in the visceral organs or the skeletal body plan could be identified and an
approach developed to explore the role of the expression of the transgene in the development
of the affected tissue.
MICROSCOPIC ANALYSIS
More detailed analysis of phenotypes entails histological methods. We generally
use standard paraformaldehyde fixation followed by paraffin embedding (described in
detail in Subheading . It is sometimes useful to embed in plastic (historesin),
which provides excellent tissue preservation and also enables lacZ to be visualized
under dark field with great sensitivity . Standard H and E stain usually serves well.
Bright-field or phase-contrast microscopy are generally adequate for visualization of
craniofacial structures, although differential interference contrast (DIC) optics provide
exceptionally good views of developing calvarial bone and the osteoblastic cells that
compose the osteogenic front (unpublished observations).
use standard paraformaldehyde fixation followed by paraffin embedding (described in
detail in Subheading . It is sometimes useful to embed in plastic (historesin),
which provides excellent tissue preservation and also enables lacZ to be visualized
under dark field with great sensitivity . Standard H and E stain usually serves well.
Bright-field or phase-contrast microscopy are generally adequate for visualization of
craniofacial structures, although differential interference contrast (DIC) optics provide
exceptionally good views of developing calvarial bone and the osteoblastic cells that
compose the osteogenic front (unpublished observations).
WHOLE MOUNT IMMUNOHISTOCHEMISTRY AND IN SITU HYBRIDIZATION
It is often important to know the sites and levels of expression of the transgene.
Hence the detection of the messenger RNA or the protein (or both) encoded by the
transgene may be useful. In addition, if the transgene is likely to alter cell fate, then
an analysis of molecular markers is likely to provide critical information on embryonic
territories and the differentiation status of cells composing an affected structure. For
these purposes—transgene expression and molecular marker analysis—both whole
mount immunohistochemistry and in situ hybridization are extremely useful
Hence the detection of the messenger RNA or the protein (or both) encoded by the
transgene may be useful. In addition, if the transgene is likely to alter cell fate, then
an analysis of molecular markers is likely to provide critical information on embryonic
territories and the differentiation status of cells composing an affected structure. For
these purposes—transgene expression and molecular marker analysis—both whole
mount immunohistochemistry and in situ hybridization are extremely useful
CELL PROLIFERATION ANALYSIS
BrdU incorporation can be used to assess the proliferation status of tissues of
transgenic mice. Embryos in utero can be labeled by injection of BrdU into pregnant
female mice; postnatal animals can be labeled by direct intraperitoneal injection of
BrdU. The quantity of BrdU and the labeling time must be tailored to the tissue of
interest. We provide a protocol below that works well for craniofacial tissues
transgenic mice. Embryos in utero can be labeled by injection of BrdU into pregnant
female mice; postnatal animals can be labeled by direct intraperitoneal injection of
BrdU. The quantity of BrdU and the labeling time must be tailored to the tissue of
interest. We provide a protocol below that works well for craniofacial tissues
CELL MIGRATION ANALYSIS
Some craniofacial tissues, particularly cranial neural crest, are highly migratory.
Therefore it may be of interest to assess the influence of a transgene on the migratory
properties of a craniofacial cell population. The vital dye, DiI, has been used extensively
to document migratory patterns of craniofacial neural crest (e.g., see ref. 17).
DiI is injected directly into individual cells in embryos ex vivo or in organ culture. The
location of the dye is then monitored in frozen sections by epifluorescence or confocal
microscopy. A recent innovation in the visualization of DiI makes use of polyethylene
glycol as an embedding medium, which preserves the location of DiI and provides
single cell resolution
Therefore it may be of interest to assess the influence of a transgene on the migratory
properties of a craniofacial cell population. The vital dye, DiI, has been used extensively
to document migratory patterns of craniofacial neural crest (e.g., see ref. 17).
DiI is injected directly into individual cells in embryos ex vivo or in organ culture. The
location of the dye is then monitored in frozen sections by epifluorescence or confocal
microscopy. A recent innovation in the visualization of DiI makes use of polyethylene
glycol as an embedding medium, which preserves the location of DiI and provides
single cell resolution
Subscribe to:
Comments (Atom)
