INVESTIGATOR REQUIREMENTS FOR GENE TARGETING PROJECTS
* A completed application
form and copy of your current ACUC approval letter prior to or along with
your purified electroporation DNA. High quality electroporation DNA should
be prepared as recommended and ~100µg total, linearized, purified DNA
provided at a concentration of 2µg/ul in TE along with an original picture
of an ethidium bromide stained gel showing 200ng of the electroporation DNA
and appropriate molecular weight markers.
* Projects will be scheduled only after project
approval and receipt of DNA. DNA is quantiated and run on a gel prior to electroporation.
A non-refundable project initiation fee will be invoiced at the beginning
of the project.
* A robust screening assay is critical for accurate
analysis of homologous recombination in selected clones. It is incumbent on
the investigator to develop a sensitive screening assay and demonstrate the
ability of probes to detect control (wild-type) DNA in 2-5 µg genomic
DNA isolated from a 96 well plate <protocol>.
96 well practice plates will be supplied upon request.
Review of initial screening results with investigator is required before
potentially targeted clones are expanded. DNA from expanded clones in a 12
well plate will be provided for targeting confirmation <protocol>
and results must be reviewed prior to scheduling blastocyst injections.
GENE TARGETING SERVICES PROVIDED BY THE LABORATORY
* The DNA concentration will be verified by
fluorimetry and run on a gel. If the DNA has an inadequate concentration or
not adequately resuspended it will be return and the project rescheduled
* Maintenance of germline competent ES cell
lines
HM-1 germline competent embryonic stem cells are maintained and routinely
used by the facility. The HM-1 ES cell line, developed by Dr. David Melton,
is a feeder-independent line isolated from E14TG2a-derived HPRT deficient
129/Ola (or new 129 nomenclature, 129P2) embryos1.
Several C57BL/6-derived lines are currently being evaluated for germline
transmission.
*
Phase I: electroporation and selection
ES cells are electroporated with the targeting vector and placed under drug
selection 48 hours later. Clones surviving selection are picked within 8-10
days into 96 well tissue culture plates, grown to confluency and frozen for
future use. Duplicate 96 well plates for DNA analysis are set up when plates
are frozen. Once cell growth on these plates reaches confluency, the plates
are rinsed with PBS and frozen plates are provided to the investigator for
isolation and analysis of genomic DNA. Should be able to isolate enough DNA
from each well for a Southern Blot. Strongly suggest that familiar with isolating
DNA from 96 well plates (method) and practice plates are available upon request.
96 well plates are available for practice. Up to 480 clones will be provided
and this can be done in 1-2 electroporations if necessary to obtain this number
of clones. Typically, 200-250 clones will be provided initially and if no
targeted clones are identified, additional clones (up to a total of 480) will
be provided for this same fee. Consultation required if no target clones are
identified.
Homologous recombination (targeting) is highly dependent on the gene or locus
being targeted and therefore, cannot be guaranteed.
* Phase II: expansion ES cell clones and blastocyst
injections
Original screening results will be reviewed with the investigator. One to
four potentially targeted clones will be thawed from the 96 well plates, expanded
and 3 vials frozen for future use. DNA from the expanded clone(s) will be
provided in a single well (per clone) of a 12-well plate for targeting confirmation.
Data is reevaluated prior to blastocyst injection. More clones can be expanded
for an additional cost.
Investigators can opt to continue with blastocyst injection and chimera production.
3-4 chimeric mice from any of 1-3 targeted clones (>50% chimerism) is guaranteed
if ES cell work is done by the TTML using standard laboratory ES cell stocks.
Germline transmission is guaranteed if ES work is done by the TTML.
* Phase III (optional): mating of chimeric males
Up to 4 chimeras will be mated to C57BL/6 females to produce F1 agouti mice.
Up to 15 agouti mice provided for screening for germline transmission of targeted
allele.
Other services include:
* Consultation services are available for targeting
vector and transgene construct design, microinjection DNA purification procedures,
genomic DNA isolation, screening analysis, breeding and ACUC issues.
* Training is available for embryo manipulation,
microinjection/microsurgery, ES cell culture and other related technical manipulations.
* Vectors currently available for general targeting
PGKneo; PGKtk; and PGKneo+PGKtk; and for conditional targeting flox/PGKneo,
loxP-2FRT-PGKneo and flox-flp/neo (flp flanked neo in forward or reverse orientation).
Cre and FLP recombinase plasmids are also being acquired for in vitro excision
of loxP- or FRT-flanked sequences, respectively. We will continue to acquire
additional vector.
* Training is available for embryo manipulation, microinjection/microsurgery, ES cell culture and other related technical manipulations.
Transgenic
Projects
Blastocyst Injections
References
1. Magin, T.M., McWhir, J., and D.W. Melton. A New Mouse Embryonic Stem Cell
Line With Good Germ Line Contribution And Gene Targeting Frequency. (1992).
Nucl Acids Res 20(14): 3795-3796.
2. Selfridge, J., Pow, A.M., McWhir, J., Magin, T.M., and D.W. Melton. Gene Targeting Using a Mouse HPRT Minigene/HPRT-Deficient Embryonic Stem Cell System: Inactivation of the Mouse ERCC-1 Gene. (1992). Somatic Cell and Molecular Genetics 18(4): 325-336.