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题名：Transposon-Mediated Transgenesis in Rats! W( [+ l2 B) d% I1 {
作者：Jang and Behringer
出处：Cold Spring Harb Protoc.（《冷泉港实验室实验方案》）1 G: f( n; E- [- X( P* l* O
年代：2007

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Protocol
Transposon-Mediated Transgenesis in Rats5 K& d4 R. u' E8 G
Chuan-Wei Jang1 and Richard R. Behringer2,3

1 Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA$ @" P' B2 l" l4 e
2 University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA

3Corresponding author (rrb@mdanderson.org)7 n2 X( ^9 U/ }! x7 H* ]: q
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INTRODUCTION  H& V2 P$ s( C3 Z  w" v% T. z

This protocol describes a method for the production of transgenic rats by coinjecting circular PiggyBac transposon-containing plasmid DNA with transposase-encoding mRNA. After fertilized eggs are collected from females, pronuclei are microinjected with the DNA and mRNA and then transferred immediately into the oviducts of pseudopregnant foster females. Using this procedure, we have obtained transformation frequencies of 33%-100% (average of ~80%). Traditional methods for generating transgenic rats (i.e., microinjection of linear DNA) yield 3%-41% transgenic founders. Thus, transposon-mediated transformation of fertilized rat eggs is an efficient alternative method to generate transgenic rats.
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MATERIALS) I$ d4 M4 i: h3 M) d6 M' t% G

Reagents
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caution 1.2% Avertin (tribromoethylalcohol), prepared in H2O9 v9 Q2 L9 g! }$ _: m
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Betadine surgical scrub( B/ G& q+ u! L/ g& K/ @# A
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caution 100% chloroform
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recipe caution Diethyl pyrocarbonate (DEPC)-treated H2O# l8 s4 O6 J1 L, Q0 X' F, W
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70% ethanol
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recipe 10 mg/mL hyaluronidase (Sigma, H3884), prepared in PBS and stored at 4°C) ^* G$ M; E6 ^  u. |4 n% ?# |
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During Step 14, this stock solution is diluted to 300 µg/mL in PLG-HEPES medium.
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recipe Injection buffer- b& @4 X9 H7 Y6 P! ?9 E% Z- U
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caution 200 µg/mL LHRH agonist (LHRHa; [des-Gly10, D-Ala6]-LH-RH ethylamide acetate hydrate) (Sigma L4513), prepared in PBS and stored at 4°C8 J* ~/ _3 [4 m0 `

mMESSAGE mMACHINE T7 ULTRA kit (Ambion)( D. Q7 t7 o8 M  m0 @

recipe caution Phenol/chloroform/isoamyl alcohol
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Plasmids:

pXL-BacII PiggyBac transposon (Drosophila Genetics Resource Center, http://dgrc.cgb.indiana.edu)
There are different versions of PiggyBac transposons. pXL-BacII PiggyBac is one of the most active versions based on tests in flies (Li et al. 2005).$ e4 j. h7 v0 I. ^9 S8 x5 _7 {

pCMV-PiggyBac transposase (Richard Behringer Laboratory)6 T  Q5 e5 ?. R9 h+ f7 R0 N
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recipe PLG medium

recipe PLG-HEPES medium2 a( R/ D9 |- i; F* @

Rats:/ L% K* R- r0 B; a

Sprague-Dawley (SD) female rats (Taconic Farms), with body weights around 150-280 g" H$ |$ A! D! C. S7 v* a

SD male rats
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Long Evans males (Taconic Farms), vasectomized' ~9 V# k1 j3 J
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Silicon or light paraffin oil

Equipment" x  K. C& o6 t  a8 l, S

Culture dishes (35 mm), sterile plastic  {+ I# W/ J( r- m9 c( T

Depression-well microscope slides, d" o, G/ v/ U$ S2 J9 @7 D

Forceps
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Incubator, preset to 37°C, 5% CO20 m0 H8 P3 |7 |

Microcentrifuge$ @3 m6 O: E7 `. ^

Microcentrifuge tubes8 S$ g( T& X" B. r# d+ b) {
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Microinjection equipment
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We use an upright Leitz Laborlux S microscope with DIC optics, Leica micromanipulators, and Eppendorf CellTram vario syringes. However, any microscope with DIC or Hoffman modulation optics and micromanipulators with syringes capable of microinjection of pronuclei of mouse fertilized eggs can be used (Nagy et al. 2003).
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Mouth pipettor

QIAGEN midiprep column
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Serafin clips
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Surgical instruments, sterile

Syringe needle (30 gauge)

VICRYL suture (4.0): }- ~0 j( D8 Q! M: R
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Warming plate (37°C) for rat recovery3 R" p' Z- a) p; X, w4 v
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Wound clips) F) m& [# I5 W1 C; i
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METHOD

Preparation of Rats8 {" L( R& m' X- V- e8 ]

In our laboratory, rats are maintained on a light/dark cycle with lights on at 7 am and off at 9 pm.
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1. Four days before setting up matings, prepare 12 adult female SD rats by synchronizing their estrus cycles using subcutaneous injection of 40 µg of LHRHa (between 9 am and 4 pm) (Filipiak and Saunders 2006).

2. On the day before pronuclear microinjections, do as follows:: d( t8 \' N# @
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      i. For fertilized egg production, breed six female SD rats to male SD rats.% r' }  E- z7 a% q, Y% |( W

      ii. Breed six SD females with vasectomized Long Evans males for use as embryo-recipient foster mothers.
      For the foster mothers, the presence of a white vaginal plug indicates mating has occurred. For the egg donors, if no plug is observed, then microscopic examination of vaginal aspirate smears for the presence of sperm is required to confirm mating.
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Preparation of Transposase mRNA

3. Synthesize polyadenylated and ARCA-capped transposase mRNA from plasmid pCMV-PiggyBac using the Ambion mMESSAGE mMACHINE T7 ULTRA kit following the manufacturer’s instructions.) Y. s  M5 U6 w* P/ U

4. Extract the mRNA solution once with phenol/chloroform/isoamyl alcohol followed once with 100% chloroform.: o1 G7 f: [3 X

5. Wash the precipitated RNA with 70% ethanol.

6. Resuspend the RNA in DEPC-treated H2O to 1 µg/µL.
We have had difficulty accurately measuring RNA concentration at this point, perhaps due to the poly-A tail. However, we have obtained satisfactory results by assuming the yield is as described in the kit instructions (20-30 µg total per reaction) and dissolving the pellet in 20 µL H2O.
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7. Aliquot the RNA into microcentrifuge tubes (~1 µL RNA/tube). Freeze the tubes at –80ºC until ready for use.1 p) F$ F& d+ }+ W. \
Because of the stickiness of the RNA solution, the actual amount transferred by a P20 micropipette may be closer to ~1.5 µL/tube. Regardless, this gives satisfactory results.
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Preparation of Transposon Plasmid
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8. Clone the gene of interest into the pXL-BacII PiggyBac transposon plasmid.( z; N1 F1 G! j- S1 s3 G' n2 \7 h: Q
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9. Purify the plasmid DNA using a QIAGEN column, and resuspend the DNA in injection buffer.9 k. ^2 Y" m2 ]# q/ U5 ?
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10. Dilute the DNA to 1 ng/µL with injection buffer.
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Collection of Fertilized Rat Eggs

11. Sacrifice ovulated and mated females around 5 pm. (This is based on a light-dark cycle of lights on at 7 am and off at 9 pm.)  B( c' x4 x9 {1 C9 J
By the time the eggs are collected, those that are fertilized should show obvious expanded pronuclei.! B8 i+ b2 l0 o

12. Dissect the oviducts, and place them in a dish of PLG-HEPES medium.
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13. Tear the oviducts with forceps or syringe needles to release the egg masses.( X$ Q% |- g# g
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14. Transfer the egg masses into PLG-HEPES medium containing hyaluronidase (300 µg/mL) to remove cumulus cells. Wash the eggs in PLG-HEPES and then in PLG medium before culturing them in PLG droplets covered with silicon oil in a culture dish at 37°C in 5% CO2.

Preparation of DNA/RNA Mixture. A7 Z/ a5 |) t: H

15. Take out one tube of transposase RNA and add 10 µL of transposon-containing plasmid DNA (circular, undigested) diluted to 1 ng/µL.* i/ A1 E  ]: x3 P4 \5 S: y5 r
The final concentrations are ~100-200 ng/µL RNA and ~0.9 ng/µL DNA.

16. Centrifuge the DNA/RNA mixture for at least 5 min at maximum speed in a microcentrifuge at 4ºC.
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Microinjection

17. Place droplets of supernatant DNA/RNA (from Step 16) and a small pool of PLG-HEPES (~5-10 µL) onto a depression-well microscope slide (see Fig. 1). Cover the drops with silicon or light paraffin oil.
The DNA/RNA droplets will be used to fill the microinjection needle.

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18. Load the fertilized eggs into the PLG-HEPES drop using a standard mouth pipette.
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19. Inject a sufficient amount of DNA/RNA into one pronucleus of the fertilized egg to see it expand.
The plasma and nuclear membranes of fertilized rat eggs are very elastic, requiring an abrupt or repetitive stabbing motion to penetrate the pronucleus.
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20. Wash the injected eggs that have survived the microinjection in PLG, and hold them at 37°C in 5% CO2. Proceed immediately to Step 21.* _6 T* W. ?& S0 L2 N: i2 I. b
The injected eggs must be transferred into the oviducts of pseudopregnant fosters immediately after microinjection.
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Transfer of Injected Eggs into the Oviducts of Foster Females# r& x4 a2 U  K& b* U# e
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21. Using a pair of forceps under light, check the vaginas of females mated with vasectomized males for the presence of a copulatory plug.
The vasectomized males do not produce sperm, precluding use of vaginal aspirates as a method to detect successful mating. The response to LHRHa varies, but usually ~50%-80% of the females are plugged.* h6 o( H. {* f7 N  x

22. Anesthetize the female recipients by an intraperitoneal injection of 1.2% Avertin at 0.2 mL/kg body weight.$ b* o2 A' C% X
We normally start with 2.4 mL and increase the dose gradually by 0.3-0.5 mL, until the rat is fully anesthetized.2 n$ N" h* P% b. i! I
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23. Shave the back, and scrub it with Betadine. Using sterilized surgical instruments, make a 1.5-cm longitudinal incision through the skin above the lumbar spine. Make a 1.0-cm transverse incision into the body wall, 1.0 cm lateral to the spinal column.' r2 ]( K5 Q# w
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24. Exteriorize the ovary, oviduct, and uterus. Secure them using a serafin clip attached to the ovarian fat pad.

25. Make a small hole in the ampulla of the oviduct using a 30-gauge syringe needle. Transfer 20-30 injected eggs into the ampulla using a mouth pipette.
Generally, we only transfer embryos into one side of each female recipient, usually the left side.
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26. Remove the serafin clip and gently place the reproductive organs back into the body cavity. Close the body wall with 4.0 VICRYL suture. Close the skin incision with wound clips.
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27. Place the females in a clean cage on a 37°C warming plate to recover.
In our hands, the females give birth 22-23 d after the surgery.
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DISCUSSION

In our hands, this procedure has been used to transform both rats and mice, using constructs of several different sizes. The efficiencies we have observed so far are shown in Table 1. In comparison, traditional methods for generating transgenic rats (i.e., microinjection of linear DNA) yield 3%-41% transgenic founders (Popova et al. 2005; Filipiak and Saunders 2006).

We have obtained transgenic mice using this method at a lower frequency (average = 24%) than what we found for transgenic rat generation. pXL-AttP/B-T3-Bhr2 contains a mouse tyrosinase minigene (Overbeek et al. 1991). Nine of the 10 transgenic rat founders generated using this plasmid were pigmented, demonstrating transgene expression. As an aside, the pigmentation pattern shows that the SD stock we obtained from Taconic Farms is homozygous for the hooded mutation. Southern analysis and preliminary breeding of the founders suggests that the transposons have integrated at multiple locations in the genome. This will complicate breeding schemes but may be useful if transgene expression is being assayed only in founder animals. Transposon-mediated transgenesis may also facilitate transgenic studies in livestock and other mammalian model organisms.$ J' c6 Q& l( y7 b% q
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ACKNOWLEDGMENTS+ T( |+ n1 N- o5 s2 r
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We thank Guy Eakin, Robert Hammer, Paul Overbeek, and Thomas Saunders for helpful advice. This research was supported by a grant from the National Institutes of Health (RR22904). DNA sequencing and veterinary animal care was supported by the NIH Cancer Center Support Grant CA16672.
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REFERENCES

Li, X., Harrell, R.A., Handler, A.M., Beam, T., Hennessy, K., and Fraser, M.J. 2005. piggyBac internal sequences are necessary for efficient transformation of target genomes. Insect Mol. Biol. 14: 17–30.[Medline]

Filipiak, W.E. and Saunders, T.L. 2006. Advances in transgenic rat production. Transgenic Res. 15: 673–686.[Medline]% E7 O7 i* q  R  m7 J
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Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. 2003. Manipulating the mouse embryo, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Popova, E., Bader, M., and Krivokharchenko, A. 2005. Strain differences in superovulatory response, embryo development and efficiency of transgenic rat production. Transgenic Res. 14: 729–738.[Medline]
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Overbeek, P.A., Aguilar-Cordova, E., Hanten, G., Schaffner, D.L., Patel, P., Lebovitz, R.M., and Lieberman, M.W. 1991. Coinjection strategy for visual identification of transgenic mice. Transgenic Res. 1: 31–37.[Medline]) d8 \* u+ Y9 M4 n4 Z4 R4 ]
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