标题: READING TIME TOPIC5 [打印本页] 作者: linxingxing 时间: 2009-5-3 20:32 标题: READING TIME TOPIC5
DNA microinjectiion' k7 _/ p$ n m2 x' R; ^0 Q4 Q
Injection of DNA for the production of transgenic mice or embryos. 7 M7 I+ X4 [8 m& u7 v
& v8 b6 a% H$ W! @3 c6 U wThe Transgenic Animal Center will inject DNA provided by the client into 200 eggs per construct. After injection, the eggs are transferred to the oviduct of pseudopregnant recipients. If a minimum of 2 founders per plasmid construct are not obtained, the center will injects an additional 100 eggs at no extra cost. If after a second injection session there are still no founders, no future microinjections will be performed. A meeting will be held between the center and the client to discuss future plans. 5 ?7 s8 ^; t' N2 Q# O' O; l9 m8 Q' m& Y1 T" j3 p( }1 k9 {6 W# [
One week after birth, we will do genotyping on mouse toes free of charge. Upon 3 weeks after birth, founder pups are separated and transferred to the client." S! H$ j0 }" k4 X$ g; y
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In the case that the client requires transgenic mouse embryos, although our successful rate is relatively high, no explicit guarantees will be made due to the lowered rate of charge. The client can opt to do additional injections on our much discounted rate to increase the chance of success. ; g- U4 G3 ] K1 y3 M {0 J0 B& M( j' q3 f4 f2 M$ `
While we guarantee at least two founders carrying all or part of the transgene, we do not guarantee expression or germline transmission. If the transgene is embryonic lethal, or DNA provided by the client does not meet requirements, the client understands that he/she is responsible for payment in full of fees associated with microinjection. There is no founder guarantee for BAC. 0 T v0 ?9 k" w$ Q! O - Y$ l% ~8 }8 \! j4 mTypical services include:. _4 }( ]7 M7 h* \
, z7 A0 y; K) C; v0 X( \! QSuperovulation of egg donors and mating with stud males maintained by the Center; 4 E+ e; x2 `) m) x7 O& t o: R( I3 l
Creation of pseudopregnant surrogate mothers by mating outbred CD1 females with vasectomized males and checking for vaginal plugs the morning of injection; 4 ?8 c* g& v; ^6 z0 k& _Harvesting of eggs from euthanized donors; 3 B3 C T% m8 n9 V
Dilution of the client' s transgene DNA with injection buffer to a concentration of around 5 ng/μl and injection of this solution into one pronucleus of each fertilized egg; 5 L3 X) a' c/ Z% pTransfer of all eggs that survive the injection process into the oviducts of pseudopregnant foster mothers; 5 U1 e) C0 p$ {7 Y% x, I8 T7 y9 f- `9 UMonitoring of foster mothers before and after birth of pups; ! z6 w8 u; l! g7 @Marking pups in each litter, sexing, and tail-cutting at about 2 weeks of age; ; C, ? K+ |1 N$ a9 W3 ^, V4 x* {Genotyping of DNA extracts from toe biopsies or yolk sac; & i6 r1 q7 j7 ^. J$ |% y' j
Weaning, identification, and transfer or shipment of transgenic pups to the client; 9 Q8 i+ H: L0 j
In case that the client requires transgenic embryos in stead of live mice, the procedure includes harvesting of embryos at client specified stage (i.e., E11.5), genotyping using yolk sac DNA, fixing of embryos and staining of embryos upon request. 7 z; I# Y6 ]/ f7 z7 v& p/ KFor DNA preparation please refer to: ! P$ w3 r5 g4 t; `4 K& c# O . [% b# n; @! Q2 xPlasmid DNA Prep requirements 5 h2 t- i' s% T, A) R( ~) u4 X! g3 d% Q) l
BAC DNA Prep requirements. ' c, I, I5 G2 I( i 1 m, \, D& ^5 o1 qFor detailed information of our services, Please refer to: 8 R# T6 T; j! e; W0 I6 k& _7 B; g- Y: G
Frequently Asked Questions.作者: linxingxing 时间: 2009-5-3 21:06
This is the instruction of DNA microinjection on the webset of Cyagen.作者: tanekay 时间: 2009-5-5 21:45
