Archived Protocol

<html xmlns:o="urn:schemas-microsoft-com:office:office" xmlns:w="urn:schemas-microsoft-com:office:word" xmlns="http://www.w3.org/TR/REC-html40"> <head> <meta http-equiv=Content-Type content="text/html; charset=iso-8859-1"> <meta name=ProgId content=Word.Document> <meta name=Generator content="Microsoft Word 9"> <meta name=Originator content="Microsoft Word 9"> <link rel=File-List href="./TropmatingNew_files/filelist.xml"> <title>X Tropicalis Mating Protocol</title>  <style>  </style> </head> <body lang=EN-US style='tab-interval:.25in' bgcolor="#ffffee" text="#000000" link="#0000FF" vlink="#0000FF" alink="#0000FF"> <div class=Section1> Grainger Lab X. tropicalis Mating Protocol<o:p></o:p> Updated June 1, 2001<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> This protocol is designed to produce the maximum number of offspring from a natural male/female mating. It uses artificial stimulation by HCG to induce maturation of gametes and mating behaviors. In general, we rest animals from two to three months between mating attempts, although males have been reused with as little as two weeks rest. The following things need to be done in the order in which they are listed for the mating to have a successful outcome.<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> <h1 style='text-align:justify'>Sexing Animals<o:p></o:p></h1> <h1 style='text-align:justify'> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p></h1> <h1 style='text-align:justify'>Sexing animals is the most difficult part of the mating process. We use the sum total of several criteria to make an educated guess as to the sex of the animals. They are:<o:p></o:p></h1> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> Cloacal protrusion: In females, the cloaca is very prominent and usually protrudes significantly outwards. In males, two small flaps of skin on either side of the cloaca make it less obviously protrusive, although it is still readily visible, being lighter than the surrounding skin in both sexes. The cloaca is best observed when the frog is in a relaxed position with its legs drawn up alongside its flank.<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> Lateral line: The dorsal stripes of the lateral line system converge to a point just anterior to the cloaca in males. In females they do not converge but remain as two distinct stripes, one on either side of the cloaca. This is not a hard and fast rule, however, and should not be used as the only criterion in determining sex.<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> Body shape: This is usually the clearest indicator of sex, but is prominent only in older animals (more than 5-6 months). Males maintain a constant body width from forelimbs to hindlimbs while females get wider posteriorly and have a characteristic, triangular shape.<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> Body size: Males are significantly smaller than females, although this size difference does not become pronounced until at least 5-6 months of age.<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> Mating stripes: Males have lighter stripes of skin on the undersides of their arms. These stripes can be seen when the animal is put into a clear tank. Males also develop dark pads on the palms of their hands when they are ready to mate, but these are often not present until after the animal has been primed with HCG.<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> Handling Animals<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> We use fish-nets to move animals between tanks. Care must be taken, however, because tropicalis are quick and lively and can easily jump out of a net. Desired animals are removed from their home tank and placed in clear 4 liter plastic containers (Rubbermaid) filled with unchlorinated well water (pH 5.8 - 6.0), called "frog water". They are kept in frog water throughout the mating procedure. Males and females are placed in separate tanks.<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> Hormone Injections<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> Hormone injections are usually done in the lab rather than in the animal room. Animals are immobilized using the "burrito" method: laboratory paper towels, soaked with frog water, are used to swaddle the animal and prevent it from moving. Then a small hole is torn in the part of the towel covering the posterior of the animal and the dorsal surface of the frog is exposed. An alternative method is to place the animal on top of some wet paper towels on the bench top and cover its eyes with the palm of one hand. This usually calms the animal enough so it can be injected without further restraint, but there is some risk of injury to the animal and/or the experimenter if it should struggle while being injected. This method is used most successfully by members of our laboratory with small hands.<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> We use two injections of HCG (Chorulon Intervet USA, 302-934-8051) to induce the frogs to mate (or to induce a female to produce eggs for in vitro fertilization or transgenesis). Male and female frogs are injected with the same amounts. The first, or priming, injection is 20 units of hormone in a volume of 0.1ml (we use the diluent supplied with the HCG). This is followed by a second, or boosting, injection of 100 units in 0.1ml. Injections are made into the dorsal lymph sac by inserting the needle just beneath the skin between the dorsal lateral line stripes (see the Cold Spring Harbor Xenopus manual for proper injection technique). After the first injection, return the frogs to their original, single sex containers and leave them in a quiet place at room temperature overnight. The priming injection should be done 24 to 48 hours before the boosting injection. After the boosting injection, the animals should be divided into male/female pairs and placed in clean frog water in a fresh bucket. The mating tanks should be put in a quiet place and covered with a cloth if possible. Amplexus should begin 3 to 4 hours after boosting and can continue for up to 6 hours. We normally rest animals between two and three months before re-mating them. <o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> A timetable for a typical pair mating:<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> 1 to 2 days prior to mating: Select and sex animals to be mated<o:p></o:p> Prime each animal with 20U HCG<o:p></o:p> Keep at room temperature in single-sex containers<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> 3 to 4 hours prior to mating: Boost each animal with 100U HCG<o:p></o:p> Place male/female pairs in mating buckets<o:p></o:p> Keep at room temperature in a quiet area<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> Mating: Observe animals periodically<o:p></o:p> Collect eggs at desired intervals<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> Egg Collection<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> X. tropicalis eggs are quite sticky and can adhere strongly to the walls of the mating bucket, as well as to the frogs themselves. If it is necessary to collect fertilized eggs at intervals, then it is best to remove them from the bucket with a wide-mouth plastic pipette. Patience is required, but the majority of eggs can be removed without causing undue disturbance to the mating pair. Eggs so removed can be cysteine treated (see below) to remove the jelly coat or left alone to develop naturally. If it is not necessary to obtain eggs at certain intervals, the best approach is to wait until it is apparent that no more eggs are being laid (or until the male releases the female). Often, the frogs themselves are encrusted with eggs and it is necessary to scrape them off with one's fingers. Then remove the animals, drain the water (most of the eggs will stick to the sides - the remainder can be caught with a piece of nytex mesh), and cysteine the eggs directly in the mating bucket.<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> Removal of the jelly coat is done with a solution of 2% cysteine (Sigma) in 1/9x MBS, pH 8.0. Swirl the eggs in the cysteine until the space between eggs (indicative of the thickness of the jelly coat) is minimized. This occurs within 1 to 2 minutes and eggs should not be left in cysteine any longer than is absolutely necessary. Eggs should then be transferred to a clean container and thoroughly washed (5 x 250ml washes with frog water) before being placed in a clean container in 1/9x MBS + gentamycin sulfate (50mg/ml). Some batches of eggs remain sticky even after cysteine treatment and, in these cases, we find it helpful to add 0.1% BSA to the MBS.<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> Early Embryo Care<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> X. tropicalis embryos can tolerate a range of temperatures from 20<span style='font-size:14.0pt;mso-bidi-font-size: 10.0pt;font-family:Symbol;mso-ascii-font-family:"Times New Roman";mso-hansi-font-family: "Times New Roman";mso-char-type:symbol;mso-symbol-font-family:Symbol'>°C to 28<span style='font-size:14.0pt;mso-bidi-font-size:10.0pt;font-family:Symbol; mso-ascii-font-family:"Times New Roman";mso-hansi-font-family:"Times New Roman"; mso-char-type:symbol;mso-symbol-font-family:Symbol'>°C. We typically keep early stage embryos at either 22<span style='font-size:14.0pt;mso-bidi-font-size:10.0pt; font-family:Symbol;mso-ascii-font-family:"Times New Roman";mso-hansi-font-family: "Times New Roman";mso-char-type:symbol;mso-symbol-font-family:Symbol'>° or 25<span style='font-size:14.0pt;mso-bidi-font-size:10.0pt;font-family:Symbol; mso-ascii-font-family:"Times New Roman";mso-hansi-font-family:"Times New Roman"; mso-char-type:symbol;mso-symbol-font-family:Symbol'>°C in 1/9x MBS + gentamycin sulfate (50mg/ml). Although embryos can survive at temperatures as low as 20<span style='font-size:14.0pt; mso-bidi-font-size:10.0pt;font-family:Symbol;mso-ascii-font-family:"Times New Roman"; mso-hansi-font-family:"Times New Roman";mso-char-type:symbol;mso-symbol-font-family: Symbol'>°C, it is not recommended because of the high frequency of gastrulation defects observed. As a rule, we keep a maximum of 100 embryos in a 100mm petri dish. Embryos should be sorted and dead ones removed immediately after cysteine treatment and again several hours later. Removal of dead or dying embryos and changing into fresh 1/9x MBS + gentamycin should be done daily for the next two days. After three days the media should be changed to 1/9x MBS without gentamycin or any other antibiotic. We have found that older tadpoles are very sensitive to antibiotics and will die quickly if left in gentamycin after three or four days of development.<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> Feeding of young tadpoles should begin after about five days of development (see Tadpole Husbandry Protocol).<o:p></o:p> <![if !supportEmptyParas]>   <![endif]> <o:p></o:p> X. tropicalis require lower salt concentrations in their media than do X. laevis. We keep them in 1/9x MBS for two or three days, then transfer them to 1/20x for a few days, and finally, after about five days, into tadpole water without any salt supplement.     <a href="#top">Back to Top</a>     </div> </body> </html>