ACRC / NCBS Mouse Genome Engineering Facility

Requisition FORM for Transgenic Mouse Production

1)Please carefully read the MGEF recommendations on how to prep and purify your “injectable” DNA destined to mouse pronuclear micro-injection. (detailed recommended protocol available on our website)

2)MGEF DNA fragment purification service request (1000 Rs per sample purification):

Yes or No?:

3)If you wish to purify the DNA yourself: Please provide 3-5g of linearized & purified DNA fragment @ (50-200ng/l in embryo water)- Please attach RECENT dated agarose gel purification / quantification gel picture of provided DNA.

4)If MGEF is purifying the DNA: please provide 30g of circular or digested endotoxin free prepared plasmid DNArecent dated gel picture of the 1ldigest test run. Make sure the DNA or digest is stored in proper conditions before it is handed over to the MGEF.

Budget Code # or funding source
Principal Investigator Institute name & lab address
Relevant IAEC project ID #
Contact Person
Email (required)
Phone # (required)

DNA Construct Information:

Construct Name
MGEF Project Name:
Design: Please attach RE plasmid map highlighting relevant promoter/enhancers / UTRs / protein cDNA / stop-polyA / specific inserted sequences or ORFs….
Reporter gene (if any):
LacZ
GFP, other, none….?
-Size of the linearized DNA fragment to be injected(in bp)
-Size of total vector (in pb)
Validated Expression data in vitro?: Yes or No?
Please explain and attach expression data
Relevant Genotyping primers / strategy
and PCR band size—
please provide gel picture of PCR Genotyping results on purified construct-

A-Mouse Strain (oocytes used for injections):

-The MGEF will routinely do injections in B6D2-F2 oocytes.

-Other genetic backgrounds are available upon request. (please do account for extra breeding time and potentially lower efficiency rates).

-If the project involves using your source of animals (males or females or both): please discuss with MGEF of the availability of these animals/ strains/ fertility efficiency/ superovulation efficiencies…ect…).

Typically the MGEF will require 6-10 proven young (less than 6months old) isolated stud males and a pool of 12-20 same age virgin females per week to be sacrificed as oocyte donors… this therefore implies a serious breeding program and scheduling as well as consequent number of cages destined only for these experiments.

B-Scientific Outline:

Please briefly justify in less then 500 words the purpose of generating this transgenic mouse line detailing the relevance to your research.

Is this requested stock or highly similar stock already available in another repository /Institution? (YES /NO)

If the requested line is already available elsewhere: please justify the need to repeat this procedure.

C-Animal Housing Information:

The MGEF will house the newly born pups for a maximum of 4 weeks after birth for Internal users and 8 week for external Users.

The subsequent housing / genotyping and screening is to be taken over by the labs themselves. For external Clients: Genotyping will done by MGEF staff at a per sample fee depending on Genotyping protocol-

Please provide the Name of the Animal Facility, vivarium# and room# where the animals are to be transferred to once the “potential transgenic founders” have reached the age of 3 or 8 weeks.

For Internal Users: please indicate the Number of additional cage space (for which SPF level?, V# and Room#) requested for the next 8-12 months of housing, breeding and experimental use of this new strain?

D-Results: The MGEF does ask that you provide the number of obtained PCR positive and Expression Positive animals. This is important for our facility statistical efficiency records: Thank You.

OTHER comments / project specifics? Please provide additional details you consider important to this project- (for transient embryo analysis- please provide embryonic stage at which embryos should be harvested / type of embryo biopsy sample to be isolated for PCR genotyping / eventual phenotypes to look for? / detailed required fixation and storage protocols of embryos ….)

NCBS/INSTEM MGEF PUBLICATION requirements

I, (Full NAME of Requestor/Investigator) ______, from (Institute, Laboratory Name)______, understands and agrees to the following terms:

1)The NCBS / INSTEM Mouse Genome Engineering Facility shall be acknowledgedin all publications describing and referring to the new mouse Strain [MGEF stock ID to be provided: ____TBD______] or Transient Embryos [______].

2)MGEF shall be informed of all Publications describing or referring to the newly generated mouse [MGEF stock ID to be provided: ____TBD______]or Transient Embryos [______].

3)The newly generated mouse stock shall be referred to as per established international nomenclature and MGEF stock references detailed below: (N/A in case of Transient Embryos)

Full Strain nomenclature: ______

Short strain ID:______

MGEF stock/accession number: ______

Initial Genetic background: ______

4)Authorship rights should also be discussed in case MGEF is involved in construct design, injection sample preparations or project strategy design.

5)It is the signing project requestor/investigator’s duty to make sure that all genetically engineered constructs/ vectors/ samples provided to MGEF and used for the generation of the newly generated mouse stock [MGEF stock ID to be provided: ______TBD______] or Transient Embryos [______].

have been obtained and used according to the legal terms and conditions of the original construct/sample provider or collaborator.

Date:Requestor/Investigator Name & Signature:

NCBS/Instem MGEF Head- Name and Signature-

Agreement Clause to add a new Laboratory Mouse Strain to the ACRC National Mouse Strain Repository and database:

I, (Full NAME of Requestor/Investigator) ______, from (Institute, Laboratory Name)______,

AGREE / DO NOT AGREE (choose one) to make the Mouse Strain (Full Scientific Strain Name)[______] (accession Number : MGEF______) , generated by NCBS MGEF services publically visible on the ACRC National Mouse Strain Repository Database. I understand and acknowledge that the Animal Care and Resource Center National Mouse Strain Repository at NCBS or I will NOT redistribute this Mouse Strain to any other requestor without my prior approval and according to the Institute’s (NCBS or INSTEM) legal MTA terms.

I also agree in making sure the newly engineered stock is cryopreserved via services provided by MGEF and stored in the Institute’s Mouse Strain CryoBank within the first 2 generations of initial Founder breeding-

Ialso acknowledge that all animal shipping / transportation procedures and logistics will be pre-arranged and financially covered by my Lab / institute and coordinated with the ACRC Veterinarian and operated to meet and respect all required animal ethics and animal welfare conditions. The NCBS ACRC/MGEF is NOT responsible for the shipments of live animals to and from external laboratories.

Date:Investigator Name & Signature:

NCBS/Instem MGEF Head- Name and Signature-

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