Ben J. Zeng
The organogenesis during development begains with a fertilized egg, and through proliferation
and subdivision into distinct groups. The differentiation and mapping of cell lineages located in
a primordia is controled by genetic patterning that involves the signalling between cells communication
and transduction within cells. The genes expressed at low levles or specificically in certain cell
tpyes, which include the key regulatory factors responsible for differentiated phenotype, developmental
progression, or cell growth regulation are largelly undiscovered. The biotechnology of transgenics and
ES cell mutagenesis is important for the gene identification and functional analysis of genomics.
Controlling the spatial and temporal expression patterns of transgenes, however is a
prerequisite for the elucidation of gene function, and producing recombinant proteins in the specifici
tissue of organism. Integration of the transgene into the host chromosome is a very rare and random
event, which makes pronuclear injection tedious, costly and inefficient. Live animales can be derived
from cultured cells (ES or adult cells) via nuclear transfer into an enucleated oocytes, or direct
injection genetic modified ES cells into a host blastocyst (chimeric offspring).
The production of transgenic animals will be greatly enhanced with the development of cultured cell
lines that remain totipotent following nuclear transfer. Cloning offers high efficiency to transgenic
research. The availability of donor cells isolated from transgenic animals that can be maintained
in culture and be frozen for storage. The more importantly is the ability to use gene targeting to
modify and select populations of cells of specific types. Nuclear transfer from cultures somatic
cells provides an alternative means of cell-mediated transgenesis.
For cloning of mice, the first step, use a fibroblast cell culture for transfection and
selection of stable expression cell line which enables to use for nuclear transfer of unfertilized
egg. The integrated gene, expression at mRNA and protein levels, be determined by PCR, northern bloting,
western blotting. The stable expression cell line be selected and established for used as nuclear donor
for nuclear transfer. The next step, transfer the somatic cell nuclei into enucleated oocytes,
use in vitro embryo culture and transportaton of embryo into the urerus of pseudopregnant recipient mice.
The PCR, Northern and Western blotting be used for analysis the transgene expression.
The Cre, a recombinase protein, can mediate intramolecular (excisive or inversional) and intermolecular
(integrative) site specific recomination between loxP sites. Therefore the Cre/LoxP system can be used for
tissue-specific removeal of a intervening stop sequence between the promoter and the coding region
of the transgene. Reporter gene assay, which include CAT, ?glactosidase, luciferase and green fluorescent
protein (GFP) etc., have contributed greatly to the study of eukaryotic gene regulated expression
both in vitro and in vivo. The Cre/LoxP combination with tetracycline responsive expression system, it
can be used for temporally and cell type specifically direct the reporting gene expression. Recently, the
RNA interfence (RNAi) can be induced within cell by transfecting plasmids that express a sequence encoding a small
hairpin RNA under the control of an RNA Polymerase III (Pol III) promoter. Therefore,
it is also possible to use RNAi for knock down gene expresssion in vivo.
The transgenesis study will be concentrated on the gene construction of Cre-Lox P system in cell type
specificial expression, cell lineages re-program by cell mediated nuclear transfer and production of chimeras
by injection of genetic modified primordial germ cells (PGC). Since the embryoes developing in an egg as
opposed to the womb, will eventually the chicken and zebrafish prove to be better test subjects than
laboratory mice for the gene function experiments. (References)