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)
Available Strains:
Vav1Cre
YAC
EED floxed
Neotg for feeder
Mir21floxed
Lsd1floxed
Rosa26LacZ Cre reporter
Rosa26ERTCre
Tel floxed
Mx1Cre
UTX floxed
SclCreERT2
Gata2 het
Ezh2 floxed
Scl floxed
EprCre
ACTFlpe
Gata1Cre
Jmjd3floxed
Rosa26BirA
ADAR1floxed
Gfi1floxed
Gata1S
Rosa26GFP CreReporter
gata2 floxed
Bcl11a floxed
Tie2 Cre
iCas9
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