How to AlterES Gene with Emory’s Transgenic Mouse Core Facility –

Gene Targeting:

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

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Quick Guide (see below for more detailed flow chart):

  1. PI submits a project request form in PPMS (if no account, create one here).
  2. PI discusses project with Core Staff in person or by phone.
  3. TMF will cut and purify the vector.
  4. TMF electroporate the vector to ES cells andstart drug selection.
  5. Colonies are picked up by the core.
  6. PI identifies positive clones via Southern.
  7. TMF expands positive clones.
  8. 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
  2. PI is responsible to build targeting vector and develop Southern screening strategy
  3. 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.
  4. PI is responsible to submit the following to TMF:
  • Vector map and screening strategy in electronic version
  • X-ray or phosphoimagerfilm show WT DNA band
  • 150 ug uncut plasmid DNA and directions of what enzyme linearizes
  • Work order form filled with required information
  1. PI order the project from PPMS
  2. TMF Core Director approves the project to PPMS
  3. TMF Core Director send a brief project outline to PI after double checked with a Core staff
  4. PI approves the project outline

Project Flow:

  1. TMF will linearize and purify the vector
  2. The vector will be electroporated into ES cells
  3. Drug selection begins for about 9 days before colonies are picked
  4. TMF will pick 200colonies
  5. Master plates are frozen after 4-5 days after colonies are picked
  6. Cell aliquot from the master plates are grown for DNA purpose
  7. 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
  8. PI is responsible for screening DNA plates provided by TMF via Southern and providing documentation with the results
  9. TMF Core Director meets with PI lab to go over the screening result
  10. 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
  11. PI lab is responsible to confirm the expanded clones via Southern and provide documentation of the results.
  12. All negative ES cell clones will be discarded after PI lab confirmed all expanded clones
  13. PI lab decides which clones will be used for microinjection with assistance from Core staff if needed
  14. TMF staff and Core Director will communicate with PI lab periodically throughout the project milestones until the project is completed

Service Rate Structure:

  1. TMF will bill PI on the month of the project initiated:
  • $6220 for HZ2.2 (129/SvEv)
  • $7460 for C57BL/6
  1. 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

1TMF will not guarantee homologous recombinant clones

2TMF will guarantee the existing Core protocol and Core SOP are followed