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Director: Paul Overbeek, Ph.D.
Email: overbeek@bcm.tmc.edu
Phone: 713-798-6421 |
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The objective of the Microinjection Core is to generate
transgenic mice by embryo microinjection for MRDDRC investigators to
use in their studies of mental retardation and developmental disabilities.
The primary function of the Microinjection Service will be to generate
transgenic mice by microinjection of recombinant DNA into mouse embryos.
The recombinant DNA will be constructed and provided by the laboratories
of the MRDDRC investigators. The core unit will purify DNA fragments
or BAC clones for microinjection, isolate the mouse embryos, microinject
the DNA into the mouse embryos, re-implant the mouse embryos into pseudopregnant
foster mothers, and supervise the husbandry of the
newborn mice. At weaning age the mice will be ear tagged for identification,
then transferred to individual investigators for further characterization.
The core lab will provide advice on construction of DNAs for microinjection,
screening of newborn mice for integration of microinjected DNA, and
characterization of gene expression in the transgenic mice.
Recombinant DNA can be introduced into the mouse
genome by microinjection of the DNA into the pronuclei of one-cell stage
mouse embryos. A fraction (usually around 20%) of the subsequent newborn
mice has been found to contain the microinjected DNA stably integrated
in their genome. The new DNA typically integrates as a tandem head-to-tail
multimer at a single site in the genome. In most cases, the DNA integrates
shortly after microinjection so that it is present in the germ line
and is stably transmitted to subsequent generations. The transgenic
DNA is typically transcribed and translated in a tissue-specific and
stage-specific pattern which is determined by regulatory sequences included
in the recombinant DNA. Consequently, transgenic mice represent an experimental
system in which it is possible to add new DNA to the genome of a mammalian
organism and then to study the consequences and/or pattern of expression
of the new genetic information.
The DNA to be microinjected can be purified after excision from standard
cloning vectors, or can be larger pieces of DNA such as intact BAC clones.
The BAC clones can be engineered by homologous recombination prior to
microinjection. The Microinjection Service will assist MRDDRC investigators
with protocols for BAC engineering and BAC DNA purification.
Transgenic mice are being used increasingly for studies of neuronal
development and neuron-specific gene expression. Experiments using transgenic
mice include: a) studies of cis-acting regulatory sequences that direct
gene expression to specific neuronal cell types, b) studies of inappropriate
synthesis of cell-surface markers that are essential for normal neuronal
morphogenesis, c) studies of neuronal maturation, cell cycle control
and apoptosis, and d) studies of nuclear inclusions and RNA binding
proteins. Such experiments contribute to a better understanding both
of normal neurological function, and of the disorders that can lead
to mental retardation.
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