READING TIME TOPIC4

linxingxing

ES Injection  }/ ^) N6 j  X) n! M! S" m* ~
In vitro Gene targeting is carried out in mouse embryonic stem (ES) cells before ES microinjection. Gene-targeted cells are injected into mouse blastocysts to make chimeric mice, whose tissues are a mosaic of cells derived from the blastocyst and cells derived from the injected ES cells. Chimeras are usually identified on the basis of coat color. Chimeric mice must be crossed with wildtype mice to determine germline transmission, meaning the ES cells can contribute to the germline of the chimera and produce offspring from an ES cell-derived gamete. On average, half of such offspring could inherit the targeted gene. 0 R- k1 h9 m/ n" Z! f
  J8 @, l% h- Y+ X5 L" Q# E: b. Y
Similar to DNA microinjection to produce transgenic mice, ES cell injection involves the superovulation and mating of blastocyst donors to obtain large numbers of embryos, and injected embryos are implanted into the oviduct of 0.5 day pseudopregnant surrogate mothers. ES cells to be injected can be supplied by the client or grown in our facility. Usually, cells are expanded from frozen stock a few days prior to injection. $ c+ L/ E; D9 V1 o5 }" I

) ]8 U, R0 r6 w& FES cell clones will be microinjected into a minimum 60-80 blastocysts per session. After injection, the blastocysts are transferred into surrogate mother and allowed to develop to term. Upon weaning 3 weeks after birth, at least 2 chimeric mice are transferred to the client. The center cannot guarantee germline transmission.
# N2 f0 F1 Q6 ~% y; n
: L4 h- h% o+ Q/ D2 F- R3 l4 g3 O9 oTypical services include:
+ z" g7 S7 x* o' E2 |1 U
  h+ z4 ~+ |6 J* K: y9 IPreparation of superovulated and mated blastocyst donors
- h# n) i' K7 `3 hCreation of pseudopregnant foster mothers by mating ICR females with vasectomized males and checking for vaginal plugs the next day ! q8 X3 c1 N5 _, M3 a
Injection of as many expanded blastocysts as possible with about a dozen to 15 ES cells each
7 K; }/ U3 U- v. L( [Transfer of injected blastocysts to the oviducts of foster mothers
" K& P3 }( Y  S8 f  F8 wMonitor foster mothers before and after birth of pups 6 q9 _! B3 F' G1 h8 _
Identify chimeric pups by coat color and determine sex
* I1 L& J/ l  T& s8 CTransfer chimeric pups to client after weaning