How to Alter ES Gene with Emory’s Transgenic Mouse Core Facility –
Gene Targeting:
PI
TMF
Vector design by
Cloning Core
Week 1
Electroporation
Week 2
Pick 200
colonies
Week 3
DNA plates on 96well-plates
Week 4
PI genotypes
a
by Southern
Week 5
Gene targeting timeline:
Week 1: set up for electroporation
Week 2: electroporation and begin selection
Week 3: pick colonies into 96 well plates
Week 4: freeze replica 96 well plates/set up replica 96 well DNA plates
Week 5: lyse 96 well plates and provide to lab for DNA analysis
a
DNA prepared from picked up colonies is screened for the presence of the transgene.
The first two lanes are controls, a positive and a negative. The next four lanes show a
negative, two positive and another negative sample in order.
Quick Guide (see below for more detailed flow chart):
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PI submits a project request form in PPMS (if no account, create one here).
PI discusses project with Core Staff in person or by phone.
TMF will cut and purify the vector.
TMF electroporate the vector to ES cells and start drug selection.
Colonies are picked up by the core.
PI identifies positive clones via Southern.
TMF expands positive clones.
PI confirms positive clones via Southern.
Detailed Guide:
Before Project Begins:
1.
PI needs to meet with the Core Director for a free consultation to go over the targeting vector design and ES cell colony screening
strategy
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PI is responsible to build targeting vector and develop Southern screening strategy
PI needs to meet with the Core Director to document the proven Southern probes work on wild type (WT) DNA blot, clearly show
WT bands.
PI is responsible to submit the following to TMF:
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Vector map and screening strategy in electronic version
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X-ray or phosphoimager film show WT DNA band
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150 ug uncut plasmid DNA and directions of what enzyme linearizes
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Work order form filled with required information
PI order the project from PPMS
TMF Core Director approves the project to PPMS
TMF Core Director send a brief project outline to PI after double checked with a Core staff
PI approves the project outline
Project Flow:
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TMF will linearize and purify the vector
The vector will be electroporated into ES cells
Drug selection begins for about 9 days before colonies are picked
TMF will pick 200 colonies
Master plates are frozen after 4-5 days after colonies are picked
Cell aliquot from the master plates are grown for DNA purpose
Duplicate DNA plates are prepared when the cells are confluence, DNA will be stored in 70% ethanol at -20oC until PI lab request
one or both set of the plates
PI is responsible for screening DNA plates provided by TMF via Southern and providing documentation with the results
TMF Core Director meets with PI lab to go over the screening result
TMF will thaw master plates after PI lab electronically informed TMF for the initial positive clones, master plates will be re-frozen
immediately after the colonies are expanded and store at -20oC
PI lab is responsible to confirm the expanded clones via Southern and provide documentation of the results.
All negative ES cell clones will be discarded after PI lab confirmed all expanded clones
PI lab decides which clones will be used for microinjection with assistance from Core staff if needed
TMF staff and Core Director will communicate with PI lab periodically throughout the project milestones until the project is
completed
Service Rate Structure:
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TMF will bill PI on the month of the project initiated:
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$6220 for HZ2.2 (129/SvEv)
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$7460 for C57BL/6
Extra fee will be billed to PI lab in the following cases:
If no positive clones identified from the first 300 clones, the core will repeat gene targeting with newly prepared plasmid supplied by
PI lab at no additional cost, however, additional fee will be charged for $1290 for each 96 clones after another 200 clones screened
$200 for each additional ES cell clones when PI requested to expand
Project Guarantee and Quality Control
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TMF will not guarantee homologous recombinant clones
TMF will guarantee the existing Core protocol and Core SOP are followed