Services provided by the Transgenic and Targeted Mutagenesis
Laboratory include:
• Consultation services: Expert consultation is available for targeting
vector and transgene construct design, microinjection DNA purification procedures,
genomic DNA isolation, screening analysis, breeding and IACUC issues.
• Training of individual investigators or their lab personnel: Training
is available for embryo manipulation, microinjection/microsurgery, ES cell
culture and other related technical manipulations.
• Access to various cloning vectors: 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
continue to acquire additional vectors and will upgrade our web site to include
vector databases with precise sequence maps.
• Access to germline competent HM1 (129/Ola) ES cells: Additional ES
lines are being acquired including a C57BL/6 line. Due to MTA agreements,
HM1 cells cannot be distributed to investigators by the facility. However,
individuals can use these cells within the facility for gene targeting experiments.
Cells will be provided on the day of electroporation. We are currently obtaining
an MTA for the use and distribution of R1 cells.
• Gene Targeting services include: ES cell maintenance, electroporation
of targeting vectors provided by the client labs and positive-negative selection
of resulting clones. Approximately 200-480 clones surviving selection are
given to investigators for identification of integrated sequences into the
ES cell genome. Targeted clones are thawed, expanded and cell pellets provided
for confirmation of a correct targeting event. Identified clones are tested
for mycoplasma and chromosomal content prior to blastocyst injection.
• Production of chimeric mice by blastocyst injection: Targeted ES
cells provided by either an individual investigator or generated in the Facility
are injected into 3.5d C57BL/6 blastocysts and transferred into the uterine
horns of pseudopregnant recipients. Generally, one or two injections/HM1-derived
clone are sufficient to generate a minimum of 3 chimeric males with greater
than 60% ES derived color coat contribution. Chimeric males can be bred into
the F1 generation by the facility as a service or transferred to the investigator.
• Production of transgenic mice by pronuclear injection: All purified
transgenic DNA received for microinjection is quantified by minifluorometry,
electrophoresed and tested for toxicity prior to injection. Acceptable DNA
is injected into approximately 150-200 fertilized single-cell embryos. Initially,
tail samples from approximately 25-30 3-week old weanlings will be provided
for analysis of transgenesis. If no founders are identified from the initial
set of injections, a consultation will be requested. If the screening assay
is determined to be accurate and adequate, a second set of injections will
be performed. If embryonic lethality is suspected after consultation, injections
for analysis by transient transgenesis could be performed if desired for an
additional fee.
• Founder screening: PCR analysis of genomic DNA for transgene integration
is not routine but is available on request and contingent on time considerations.
Investigators must supply primers and positive control templates.
More information on specific services and investigator
requirements for projects can be found below:
Transgenic
Projects
Blastocyst Injections
Targeted Mutagenesis Project