Tetraploid Embryo Complementation

The method of tetraploid embryonic complementation is used to verify the full pluripotent potential of embryonic stem (ES) and in vitro reprogrammed pluripotent stem (iPS) cells. The two blastomeres of an embryo at the two-cell stage are induced, by electrofusion, to merge into one that will then have a tetraploid genome and will continue to divide. The resulting embryonic tetraploid cells are no longer able to form a living mouse, but they differentiate successfully into trophoblast cells, which later form the placenta. When ESCs or iPS cells are injected into the otherwise morphologically indistinguishable embryo, an all ES-derived embryo will develop from the injected cells, demonstrating their pluripotent potential.

Blastocyst injections include injection of 45-50 blastocysts. A single clone will be injected in a day. It may be possible to inject more embryos – if available – for an increased fee.

FEES
	WI	MIT	External Academic
Injection of ESCs or iPSCs – Charge per Injection Day
Into Tetraploid Blastocysts (all-ESC-derived mice)	$2,000	$2,500	$3,500
Additional Costs
Housing	$1.5/cage/day	$1.5cage/day	$2 cage/day
Additional Injections After Deliverables Reached	$750/day	$1,000	$1,500
Other Costs (Tail biopsies, Pathogen tests, shipping to external labs, etc)	variable	variable	variable

Figure: Example of a useful application for 4n complementation

(A) The accelerated production of homozygous mutant offspring can be achieved by isolating 39,X0 subclones from targeted 40,XY ES cell lines and producing ES cell–derived males and females by tetraploid embryo complementation. These heterozygous mice can then be immediately intercrossed to produce homozygous mutant offspring, eliminating independent segregation of alleles and considerably shortening the time required to generate experimental animals. (B) Standard production of mutant mice from heterozygous ES cells necessitates the generation of chimeric founder animals by introducing targeted male ES cells into diploid blastocysts. Chimeric founders must then be outcrossed to fix the mutation in the male and female germline. These male and female heterozygous offspring are finally intercrossed to produce homozygous mutant progeny.

Figure taken from: Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation.

Eggan K, Rode A, Jentsch I, Samuel C, Hennek T, Tintrup H, Zevnik B, Erwin J, Loring J, Jackson-Grusby L, Speicher MR, Kuehn R, Jaenisch R. Nat Biotechnol. 2002 May;20(5):455-9. doi: 10.1038/nbt0502-455. PMID: 11981557