Mouse Embryonic Stem (ES) cells and Gene targeting Core

 

Since 1995, our core facility has been successfully generating more than >300 gene knockout/knockin lines for analysis gene function in hematopoiesis and others.

 

Orkin SH, Zon LI. Cell. 2008 Feb 22;132(4):631-44. The gene knockout of Gata1, Gata2, Tel, FOG-1, Gfi-1, Gfi-1b EKLF, Scl/Tal1 in this figure, were performed by this core.

This core facility provides resources for use and manipulation of mouse ES cells, and generates gene modified animal models for in vivo gene function analysis. In addition, the core provides advice, training and support for members of the center whose research requires mouse engineering in the analysis of hematopoiesis (both locally and elsewhere).

 

 

Basic Service

Generation of gene targeted mice by blastocyst injection

We only inject >80% normal karyotype ES cell lines for germline transmission purposes. For 129 ES cells (Cj7, Cj9, TCL), we will inject 2~3 independent targeted ES clones into C57Bl blastocysts.

For C57Bl ES cells (e.g. EUCOMM), we will inject into Balb/c or FVB blastocyts.

We use CD-1 pseudo-pregnant females for surrogate mothers.

 

Transgenic mice generation by pronuclear injection

We perform pronuclear microinjection with plasmid DNA fragment or BAC DNA.

Plasmid DNA fragment is isolated and purified by the core (plasmid DNA is prepared by investigators with CsCl3 prep).

BAC DNA is prepared and purified by investigators.

For embryo donors, we use C57Bl or FVB. Average 150~200 eggs will be harvested and injected. We inject one construct per day.

We use CD-1 pseudo-pregnant females for surrogate mothers.

 

Mouse ES cell electroporation, selection and DNA purification

We use cJ7, cJ9 ES cells (from 129 strain) for regular ZAP. F1 ES cells (V6.5) are also available for tetraploid complementation.

Purified plasmid DNA (from investigator) will be used for ZAP. Subsequently we will pick up ~200 clones after the suitable drug selection. We will provide 12 well amount of ES DNA for genotype.

 

Additional Services

Consultation of gene targeting/transgenic projects

 

Cells:

Training of ES cell manipulation

ES cell cassette deletion (drug selection cassette) with cre/flp recombinase cre/flp recombinase plasmid are supplied by investigators

Generation of null ES cells by selection drug step-up methods

Karyotyping

Derivation of ES cells from embryos

Derivation of iPS cells from fibroblast

Generation of mouse embryonic feeder (MEF) cells

 

Mouse:

Training and support of mouse handling/numbering/colony maintenance/ injections/surgeries

Maintenance and supply of genetically modified mouse model, such as various cre lines, cre reporter lines.

Support of embryo manipulation/dissection for phenotype analysis

Generation of teratomas

Tetraploid complementation

In vitro fertilization (IVF)

Derivation of strains of interest by embryo transfer

Embryo cryopreservation

Bioimaging Xenogen imaging

(Ultrasound imaging)

 

Personnel/Contact information

Director:

Yuko Fujiwara fujiwara@bloodgroup.tch.harvard.edu

617-919-2097

 

ES cell division:

Frank Godinho godinho@bloodgroup.tch.harvard.edu

617-919-2052

 

Mouse manipulation division:

Minh Nguyen nguyen@bloodgroup.tch.harvard.edu

617-919-2050

 

Service request forms

Blastocyst injection

Transgenic production

ES cell derivation

Embryo cryopreservation