Transgenesis in zebrafish and mice
Methods
The most efficient method of
introducing DNA to vertebrate eggs is microinjection. Linear DNA is injected into
fertilised oocytes at the one cell stage before cleavage divisions begin.
Zebrafish ooctes are very yolky making it difficult to see the nucleus so DNA
is injected at high concentration into the cytoplasm. Mouse oocytes are
relatively yolk free making it possible to inject directly into the nucleus
(and use lower concentrations of DNA). In the nucleus the injected DNA can
integrate into the fish or mouse chromosomes by a mechanism which is not fully
understood. Integration sites are random and bear no relationship to the
sequence injected (the transgene). Often multiple copies of the injected
sequence insert together at the same site and rearrangement or deletion of the
sequence can occur. Another problem is that integration may not take place
immediately and, particularly in the rapidly dividing zebrafish embryo, can
happen after the egg has divided. In these cases not all cells of the embryo
will carry the insertion - the embryo and eventually the adult is mosaic.
Mosaicism can include the germ line in which case the potential founder
animal may not be able to transmit the transgene to offspring to set up a
stable line of transgenics. Founders must be outcrossed to normal animals and
the F1 offspring tested (e.g. by PCR analysis of non-essential tissue, usually
tail).
Applications
Transgenic animals have both
research and commercial/biomedical applications:-
Much research with transgenic
animals is focused on the identification of stage and tissue specific
regulatory sequences. Some examples of this are given in the later half of the
lecture.
Regulatory sequences can be
identified by looking at the expression of transgenes in which segments of the
genomic sequence flanking a gene have been cloned adjacent to a reporter gene.
Reporter genes produce proteins the expression of which can easily be
detected.. The most common are b-galactosidase, activity of which
converts the colourless substrate X-gal to a blue compound and Green Flourescent
Protein (GFP). This is derived from jellyfish and fluoresces under UV
illumination. GFP flourescence can be observed in living transgenic animals, to
stain with X-gal animals have to be killed and fixed. Transgenes which
successfully recapitulate the cell or tissue specific expression of the
endogenous gene can be used to mark those tissues in subsequent experiments.
One important
point about using transgenics to identify regulatory elements. Transgenes can
become rearranged, or insert at locations where they are inactivated (e.g.
heterochromatin) or come under the control of endogenous regulatory elements
(position effects). It is, therefore, essential to check the expression of any
transgene in several different lines before drawing conclusions about the
regulatory effects of any sequences included.
Once cell stage
or tissue specific regulatory elements have been isolated they can be used to
drive the expression of genes other than the one from which they were
derived. Transgenes can also be
used to supplement the expression of endogenous genes, simulating the effects
of chromosomal duplications. Transgenes which mimic the expression of
endogenous genes can also be crossed into a mutant background to see if they
can rescue the effects of the mutant (functional complementation).
Transgenic mice are used to
model diseases. Mice which overexpress parts of the a-amyloid
protein precursor show Alzheimers-like symptoms and are used to test the
effects of drugs that could alleviate this condition. Transgenic mice can also
be used to model the effects of overexpression of myelin proteins observed in
congenital neurological disorders such as Charcot Marie-Tooth disease.
Mice with transgenes coupled to
the b-lactoglobulin promoter express the
transgenes in their milk. This promoter has been used to make transgenic sheep
which produce a-antitrypsin (used to treat cystic
fibrosis) in their milk - sheep being easier to milk on a commercial scale than
mice.
More controversial is the
recent development of transgenic salmon with added growth factor. There are
arguments against the licensing of such fish for general consumption on
environmental, safety and welfare grounds.
Examples
Checking the zebrafish a-actin promoter for muscle specific activity
a-actin is restricted to skeletal muscle. A 3.6kb
ftragment from the 5' end of the fish a-actin gene was fused to GFP coding
sequences and injected into eggs at the 1-cell stage.
10% of injected fish which
expressed GFP were able to to transmit the transgene. GFP expression was
restricted to skeletal muscle in most (but not all) lines.
Similar GFP lines have been
generated using promoter sequences from neural and glial specific genes. These
lines are extremely useful to developmental biologists as they allow the
development of these cells to be followed in living embryos, including mutant
embryos.
Identifying conserved
floorplate specific regulatory elements of the Shh gene
Shh is initially expressed in
the floor plate and later in other tissues such as the limb buds. The pattern
is similar in all vertebrates. Promoter sequences fused to b-galactosidase coding sequences do not express b-galactosidase in floor plate. Adding a genomic
segment covering introns 1 and 2 of the gene produces transgenic fish with
mosaic expression of b-galactosidase in floor plate cells. The
same construct injected into mouse eggs also shows floor plate specific
expression. Comparing the sequences of zebrafish and mouse introns 1 and 2
allows the region driving this floor plate expression to be narrowed down even
further.
Mapping long range
regulatory elements of the Mouse/human Pax6 (Aniridia) gene
Pax6 is a highly conserved
transcription factor with a complex and dynamic expression pattern in the
developing eye brain, olfactory system
spinal cord and pancreas. In humans mutations in PAX6 cause the dominantly
inherited eye defects Aniridia and Peterís anomaly and may also be associated
with some cases of mental retardation.
Pax6 is a large gene with 13
exons covering a 22 kb region. The ATG lies in exon 4, there is one
alternatively spliced exon (5a) and two promoters, P0 and P1. Standard
transgenic analysis of the promoter region and the more upstream introns has
uncoverd three regions (EE, ENN and NRE) with enhancer activity in the lens and
pancreas (EE), the forebrain, hindbrain and spinal cord (ENN) and peripheral
retina(NRE).
Advantage has been
taken of the availability of the complete genome sequences for humans, mice and
fugu fish to identify conserved non-coding regions downstream of Pax6. Two regions show both hich
levels of sequence conservation and enhancer activity in transgenic assays.
HS234 is a 4.5kb region >150 kb downstream of the Pax6 P0 promoter which
drives transgene expression in early eye development. C117 Box123 is a 2.9 kb
region ~77kb downstream of the Pax6 P0 promoter which drives transgene
expression in the olfactory system, central retina and forebrain.
Interestingly, transgenes made using the Box123 region often exhibit ectopic
expression in the midbrain. This result suggests that enhancers that drive
midbrain expression are present in this region, but that Pax6 expression in the midbrain
is normally inhibited by suppressor elements, which lie elsewhere in the
genome.
References
Gene regulation in “Molecular
Cell Biology” Chapter 7 pp 276-286
Transgenic animals
and food production