Screening Strategy for DATE

/ The University of Michigan
Transgenic Animal Model Core
Sally A. Camper, Ph.D.
Linda C. Samuelson, Ph.D.
Thomas L. Saunders, Ph.D. / 2560 MSRB II, SPC 5674
1150 W. Medical Center Drive
Ann Arbor, MI 48109-5674
Voice: (734) 647-2910
FAX: (734) 936-2622
http://www.med.umich.edu/tamc/

Screening Strategy for DATE:

Investigator: phone/email:

Lab Contact: phone/email:

1. Attach restriction maps of the endogenous gene, targeting vector, and targeted chromosome after homologous recombination. Indicate DNA fragments that will be used as probes on Southern blots, pertinent restriction enzymes, and PCR primers (if applicable).

2. How many Kb of DNA will be deleted from the chromosome by homologous recombination with the targeting vector?

3. Please complete this table:

Predicted DNA Fragment Sizes (Kb) on Southern Blot

Restriction / 5’ Probe
Enzyme / Endogenous Gene / Targeted Gene / Random Integrant
Restriction / 3’ Probe
Enzyme / Endogenous Gene / Targeted Gene / Random Integrant

4. Please provide film showing:

5’ Probe - Southern Blot Data on 2-5 µg of ES cell DNA

3’ Probe - Southern Blot Data on 2-5 µg of ES cell DNA

Targeting Vector for DATE:

Investigator: phone/email:

Lab Contact: phone/email:

5. Will a PCR screen be used? � yes � no

If so, please provide results that demonstrate the screen is sensitive at the single copy level. To achieve this, use a positive control construct which includes sequences recognized by the screening primers. The size of its PCR product should be noticeably different from the size of the product generated by the targeted gene. To produce a single copy template, spike 10 µg of mouse DNA with 2 pg of plasmid DNA for every Kb of the control construct.

Attach Restriction Map of Vector

genetic background of mouse providing arms of homology

length of homologous arms: 5’ 3’:

were the arms of homology generated by PCR? yes no

junction fragments sequenced? � yes � no

evidence that 5’ arm is in correct orientation � yes � no

evidence that 3’ arm is in correct orientation � yes � no

evidence that single bp mutations (if any) are present � yes � no

demonstrate screen for single bp mutations (if any) � yes � no

demonstrate that loxP sites are in the correct locations (if any) � yes � no

demonstrate that loxP sites are in the correct orientation (if any) � yes � no

how many base pairs are between the distal loxP site (if any) and the neo cassette?

demonstrate that ES cells containing an HSV-TK cassette � yes � no

will not be used to generate chimeras

will negative selection be used? � HSV-TK � DT-A � Other

If Other, please provide map and description of selection cassette.

Targeting Vector for DATE:

Investigator: phone/email:

Lab Contact: phone/email:

what is source of neo cassette? � pPNT � pNZ-TK2 � pMC1NEO � Other

If Other, please provide map and description of neo cassette source.

what restriction site will be used to linearize targeting vector?

what size is the targeting vector after linearization?

Attach the pertinent paperwork:

for R1 ES cells: copy of approved material transfer agreement: � yes � no

for CJ7 cells: copy of letter agreeing to acknowledge Tom Gridley: � yes � no

for pPNT: copy of letter requesting use of vector: � yes � no

for pNZ-TK2: copy of correspondence permitting use of vector: � yes � no

Other agreements (DT-A, GFP, IRES, lacZ, etc.)?

UCUCA Approval Number

ACKNOWLEDGMENT INFORMATION

It is important to document use of the Transgenic Core by investigators in publications. This facilitates efforts to obtain external funding for the Transgenic Core. Please include the following information in your acknowledgments if you are member of any of the research centers listed below.

“The Transgenic Animal Model Core of the University of Michigan is supported by ... (insert Centers checked below).

Cancer Center

The University of Michigan Cancer Center, NIH grant number CA46592.

Arthritis Center Members

The University of Michigan Multipurpose Arthritis Center, NIH grant number AR20557.

Center for Organogenesis

The University of Michigan Center for Organogenesis.

Diabetes Research and Training Center

The University of Michigan Diabetes Research and Training Center, NIH grant K20572.

X Michigan Animal Models Consortium

The Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor support the Michigan Animal Models Consortium, MEDC grant 085P1000815.

/ The University of Michigan
Transgenic Animal Model Core
Sally A. Camper, Ph.D.
Linda C. Samuelson, Ph.D.
Thomas L. Saunders, Ph.D. / 2560 MSRB II, Box 0674
1150 W. Medical Center Drive
Ann Arbor, MI 48109-0674
Voice: (734) 647-2910
FAX: (734) 936-2622
http://www.med.umich.edu/tamc/

Production of Gene Targeted Embryonic Stem Cell Clones

Statement of Understanding of the Nature of the Gene Targeting Service

For production of gene targeted embryonic stem cells in collaboration with the Transgenic Core I am responsible for the following tasks:

Restriction map the endogenous gene.

Establish a robust, reliable, reproducible screen that discriminates between wild type and targeted alleles.

Provide experimental data demonstrating that the Southern blot probes will confirm the genomic structure of targeted ES cell clones.

Clone the gene targeting vector.

Purify gene targeting vector DNA for electroporation according to the protocol supplied by the Transgenic Core.

Analyze genomic DNA from as many as 480 ES cell clones for homologous recombination with the targeting vector.

Timely analysis is important because of the limited life span of ES cells cryopreserved at -80°C.

Review the screening data with the Transgenic Core prior to expansion of cryopreserved clones.

Although 480 clones should contain multiple targeted clones, I accept that the Transgenic Core can not guarantee that targeted clones will be produced. If no targeted clones result, we will reconsider the design of the experiment. This is because of the inherent variability in homologous recombination in embryonic stem cells. I understand that it is expected that the expertise and effort invested in this project by Elizabeth Hughes and Thomas Saunders will be recognized appropriately. I agree to pay the $4,000 recharge for this service.

The Transgenic Core will undertake to produce gene targeted embryonic stem cells. The Core will provide/perform the following information/procedures:

Prepare an experimental time line for planning purposes.

Electroporate the targeting vector into ___ ES cells.

Will pick 480 electroporated ES cell clones (five 96-well plates).

Each plate of the five 96-well plates will be split into three.

Two plates will be cryopreserved in independent -80°C freezers.

One plate will be grown and split into two replicates for DNA preparation.

Prepare DNA from replicate 96-well plates (ten plates total, two replicates of each of the five plates of clones).

Deliver the DNA plates to the investigator for screening.

Authorized Signature Date

Core Representative Signature Date

Production of Gene Targeted Embryonic Stem Cell Clones

Statement of Understanding of the Nature of the Gene Targeting Service

For production of gene targeted embryonic stem cells in collaboration with the Transgenic Core I am responsible for the following tasks:

Restriction map the endogenous gene.

Establish a robust, reliable, reproducible screen that discriminates between wild type and targeted alleles.

Provide experimental data demonstrating that the Southern blot probes will confirm the genomic structure of targeted ES cell clones.

Clone the gene targeting vector.

Purify gene targeting vector DNA for electroporation according to the protocol supplied by the Transgenic Core.

Analyze genomic DNA from as many as 480 ES cell clones for homologous recombination with the targeting vector.

Timely analysis is important because of the limited life span of ES cells cryopreserved at -80°C.

Review the screening data with the Transgenic Core prior to expansion of cryopreserved clones.

The Transgenic Core will undertake to produce gene targeted embryonic stem cells. The Core will provide/perform the following information/procedures:

Prepare an experimental time line for planning purposes.

Electroporate the targeting vector into ___ ES cells.

Will pick 480 electroporated ES cell clones (five 96-well plates).

Each plate of the five 96-well plates will be split into three.

Two plates will be cryopreserved in independent -80°C freezers.

One plate will be grown and split into two replicates for DNA preparation.

Prepare DNA from replicate 96-well plates (ten plates total, two replicates of each of the five plates of clones).

Deliver the DNA plates to the investigator for screening.

Authorized Signature Date

Core Representative Signature Date

Billing Information

Project Name Date

Lab Contact Phone Number

Preferred form of contact:

email address FAX Number

P.I. Name

Authorized Signer: Authorized Signature:

Phone Number

Account Number Billing Department

Grant Title:

Funding Agency: Grant Number:

Center Membership:

Cancer Center Arthritis Center Center for Organogenesis

DRTC Gut Peptide Center Biomedical Engineering

/ The University of Michigan
Transgenic Animal Model Core
Sally A. Camper, Ph.D.
Linda C. Samuelson, Ph.D.
Thomas L. Saunders, Ph.D. / 2560 MSRB II, Box 0674
1150 W. Medical Center Drive
Ann Arbor, MI 48109-0674
TEL: (734) 647-2910
FAX: (734) 936-2622
http://www.med.umich.edu/tamc/

Purification of Gene Targeting Vector DNA for Electroporation

1. Purify plasmid from bacteria.

We recommend the Qiagen EndoFree Plasmid Maxi kit for the purification of the targeting vector plasmid from bacteria. Please follow the directions in the kit. Electroporation of Qiagen purified DNA has been used successfully by a number of labs. Alternatively, plasmid DNA can be purified by CsCl banding.

2. Linearize 200 µg of plasmid DNA with the appropriate restriction enzyme digest.

Run a DNA on a minigel to verify that digestion is complete. Extract the DNA with phenol-chloroform, then with chloroform and precipitate by adding NaCl and ethanol. Resuspend the DNA in TE (10 mM Tris-HCl, pH 8.0, 1.0 mM EDTA) at 1 mg/ml and deliver it to the Transgenic Core for electroporation.

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Please provide the following information when you submit the targeting vector.

Name of Targeting Vector

Size of linearized Vector

DNA Resuspended in:

DNA Concentration: Date:

Contact Person: phone/email: