Name: rat mammary epithelial cell transplantation into the interscapular white fat pad
Uploaded: 2020-03-04T13:00:00
Description: Watch this Scientific Journal Video about Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad at JoVE.com

This work was funded by the Hollings Cancer Center&#39;s Cancer Center Support Grant P30 CA138313 pilot research funding from the National Institutes of Health (<a href="https://www.nih.gov/" target="_blank">https://www.nih.gov/</a>), and funds from the Department of Pathology &#38; Laboratory Medicine at the Medical University of South Carolina. We would like to thank Marijne Smiths for recording the interview statements.

All animals were housed and maintained in an AAALAC-approved facility, and experiments described in this protocol were approved by the MUSC Institutional Animal Care &#38; Use Committee (IACUC). Animals for use in reciprocal transplantation should be an inbred or isogenic strain, with congenic status preferred or backcrossed for at least 6 generations.
1. Harvesting donor rat mammary gland epithelium
<ol>
	<li>Determine the number of donor rats needed for transplantation. 
	NOTE: Genera...

This protocol describes a mammary epithelial cell transplantation technique optimized for working with rats. Isolated mammary epithelial organoids from donor rats (3-5 weeks of age) are grafted into the interscapular white fat pad of recipient rats (also 3-5 weeks of age). Results can be interpreted as little as 4-6 weeks later, using light microscopy to examine the grafted tissue; however, the optimal amount of time between transplantation and sacrifice must be determined prior to implementing a full experiment. If too ...

Postnatal mammary gland development and ductal morphogenesis are processes largely influenced by hormonal signaling at the onset of puberty. In mice and rats, commonly used model organisms of mammary gland biology, this process begins around 3 weeks of age, where rapid proliferation and differentiation result in the formation of the mature parenchyma. The mature mammary gland can undergo numerous rounds of expansion and involution, a property that has been under investigation since the early 20th century. Within the context of hyperproliferation and cancer development, mammary gland transplantation techniques were developed in the 1950s1, and enhanced by the quantitative methodology contributed by Gould et al. in 19772,3,4. Refinement of the transplantation technique in rodents has contributed to major advances in understanding normal mammary gland biology that are still widely used to study the effect of various treatments and genetic manipulation on normal mammary gland development and disease states.
Many hypotheses have been generated and subsequently tested using mammary gland transplantation, first described by DeOme et al. in 19591. Experiments across several decades showed the propensity of ductal tissue excised from donor mammary glands to repopulate the entire fat pad5,6,7 and indicated that a critical component of mammary gland development resides in these epithelial structures. Later studies in mice showed that a single mammary stem cell can repopulate a cleared fat pad and contributed to the discovery of a single, common progenitor of basal and luminal mammary epithelial cells8,9,10. In line with these conclusions, it has been suggested that transplantation increases the pool of cells with multilineage-repopulating potential as a result of plasticity, allowing the grafted cells to grow a functional mammary gland7,10,11,12,13. Importantly, the use of transplantation techniques in rodents overcomes the limitations of cell culture-induced abnormalities14 and often provides results in just a matter of weeks.
While the procedure was originally described in the context of preneoplastic lesions in mice, it was soon expanded to rats and used in conjunction with the carcinogen treatment to establish multiplicity as a measure of cancer susceptibility15, but the popularity of transplantation techniques has followed the development of genetic tools for each species. Although mouse studies incorporating transplantation have contributed many translational findings, the parenchyma of the rat mammary gland resembles the human more closely16,17 and offers distinct advantages for studying estrogen receptor-positive (ER+) breast cancer. Mammary tumors are inducible in both species, but they differ in terms of hormone sensitivity and gene expression profiles. A primary difference is that rat mammary tumors express and depend on the function of ovarian and pituitary hormone receptors, namely, estrogen and progesterone (PR), similar to the luminal-A subtype of human breast cancer. Indeed, mammary epithelial cell transplantation, as described in this protocol, has been used to study genetic variants involved in breast cancer and determine the cellular autonomy of effects on mammary epithelial cells18.
In addition to the tumor biology, the ductal epithelium of the normal rat mammary gland exhibits a higher level of branching and is flanked by a thicker layer of stroma than the mouse. The importance of the stroma is well-documented in mammary epithelial transplantation studies. Mammary epithelium must interact with fatty stroma, and ideally its own mesenchyme, to undergo its characteristic morphogenesis19,20. Grafting tissue into a recipient mammary gland provides an optimal environment; however, the presence of endogenous epithelium can interfere with results. Preclearing the mammary gland of endogenous epithelium is commonly performed in mouse transplantation assays and requires surgical excision of endogenous mammary tissue and/or removal of the nipple1,21,22. Although possible, preclearing the mammary epithelium in post-weanling rats is not as widely-performed, mainly due to the ineffectiveness of clearing the growing mammary tree in post-weanling rats. Since it has been shown that regions of adipose tissue elsewhere in the body could support the growth of transplanted mammary epithelium21,23,24, the process of preclearing can be easily avoided in rats by grafting tissue into the interscapular white fat pad.
The transplantation method described in this paper involves the injection of enzymatically dissociated mammary gland organoids (fragments of mammary ductal epithelium and other cells types capable of morphogenesis) or monodispersed cells into the interscapular fat pad in inbred, isogenic or congenic strains of laboratory rats2. Because the interscapular fat pad is normally devoid of mammary tissue, it provides a suitable environment for multiple transplantation sites without the need to pre-clear endogenous epithelium. As a result, the host animal&#39;s endogenous, abdominal-inguinal mammary glands are not subject to surgical manipulation, develop normally, and cannot interfere with interpretation of results. Additionally, the intact mammary glands can be used for comparison to evaluate host versus donor effects on the mammary epithelium development and tumorigenesis18,25. Although repopulation of the mammary gland from a single stem cell is available for mice, it has not yet been developed for rat, mainly due to the lack of availability of antibodies to select for rat mammary stem cells25,26,27. Despite this, transplantation of monodispersed mammary epithelial cells to quantify repopulating potential can be successfully performed, and those cells will develop normally when grafted into the appropriate framework2,3,4. While organoids are good for many purposes, monodispersed cells are required for quantitative applications, for example, to determine the number of mammary epithelial cells required for the cancer initiation following ionizing radiation treatment28 or for comparing characteristics of flow cytometrically selected mammary epithelial cell populations29.
To date, the procedure described here is the most robust method of performing mammary gland transplantation in the rat with an overall goal of studying mammary gland development and mechanisms underlying breast cancer development. Often, the donor and/or recipient animals are exposed to different variables before, during, or after the epithelial transplantation. Examples include single gene studies involving chemical carcinogenesis30, radiation28,31,32, genetic manipulation of host/donor genome18, and hormonal manipulation12. A major advantage of the enzymatic dissociation described in this protocol is the opportunity to isolate epithelial organoids or monodispersed cells for complementary experiments involving flow cytometry, 3-D culture, gene editing, and more. Future applications of this technique will include additional manipulation of donor and/or host tissue with genetic engineering. For example, donor cells can be genetically altered ex vivo at any chosen genomic locus using the CRISPR-Cas9 gene editing system. Similarly, recipient rats can also be genetically altered to study the interaction between donor and recipient engineered genetic factors.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#181;M syringe filters</td>
			<td>Fisher Scientific</td>
			<td>09-715G</td>
			<td>sterile-filtering collagenase digestion media</td>
		</tr>
		<tr>
			<td>1.5 - 2.0 mL microcentrifuge tubes (sterile)</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td>containing resuspended cells and/or brain homogenate mixture</td>
		</tr>
		<tr>
			<td>100 &#181;M cell strainers</td>
			<td>Corning</td>
			<td>431752</td>
			<td>filtering brain homogenate</td>
		</tr>
		<tr>
			<td>100 uL gastight syringes with 25 gauge needles</td>
			<td>Hamilton</td>
			<td>81001 &#38; 90525</td>
			<td>For injecting graft mixture into recipient animals (1 per donor genotype/condition)</td>
		</tr>
		<tr>
			<td>1000 uL pipette tips + pipette</td>
			<td>-</td>
			<td>-</td>
			<td>transferring cells/mixtures/tissue</td>
		</tr>
		<tr>
			<td>15 mL polypropylene tube</td>
			<td>Falcon (Corning)</td>
			<td>352196</td>
			<td>brain homogenate mixture storage, or cell : homogenate mixture for transplantation</td>
		</tr>
		<tr>
			<td>40 &#181;M cell strainers</td>
			<td>Corning</td>
			<td>431750</td>
			<td>filtering organoids after washing the cell pellet</td>
		</tr>
		<tr>
			<td>50 mL polypropylene tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-6</td>
			<td>for collagenase digestion of donor mammary gland tissue</td>
		</tr>
		<tr>
			<td>60 mm dishes</td>
			<td>Thermo Scientific</td>
			<td>130181</td>
			<td>for mincing tissue</td>
		</tr>
		<tr>
			<td>Alum Potassium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>243361/237086</td>
			<td>staining mammary gland whole mount slides</td>
		</tr>
		<tr>
			<td>Anesthesia vaporizer for veterinary use</td>
			<td>-</td>
			<td>-</td>
			<td>follow institutional protocol</td>
		</tr>
		<tr>
			<td>Beta-dine or iodine</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass culture tube for homogenization</td>
			<td>Fisher Scientific</td>
			<td>14-961-26</td>
			<td>for homogenization of brain (use appropriate tube for homogenizer)</td>
		</tr>
		<tr>
			<td>Carmine</td>
			<td>Sigma-Aldrich</td>
			<td>C6152/1022</td>
			<td>staining mammary gland whole mount slides</td>
		</tr>
		<tr>
			<td>Cell counting apparatus</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean animal cages for recovery</td>
			<td>-</td>
			<td>-</td>
			<td>follow institutional protocol</td>
		</tr>
		<tr>
			<td>Collagenase Type 3</td>
			<td>Worthington Biochemical Corp.</td>
			<td>LS004183</td>
			<td>enzymatic digestion of minced mammary gland tissue from donor rats</td>
		</tr>
		<tr>
			<td>deionized water</td>
			<td>-</td>
			<td>-</td>
			<td>for chemical solutions</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>GIBCO</td>
			<td>11320033</td>
			<td>for mincing tissue, collagenase digestion media and resuspending epithelial cell mixtures</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>-</td>
			<td>-</td>
			<td>monodispersion mixture</td>
		</tr>
		<tr>
			<td>Ethanol, 200 Proof</td>
			<td>Decon Labs</td>
			<td>2705/2701</td>
			<td>mammary whole mount slide fixative, mammary whole mount slide washes, cleaning surgical incision sites (diluted)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>-</td>
			<td>inactivation solution</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher Scientific</td>
			<td>A38-212</td>
			<td>use for mammary whole mount slide fixative (1:4 glacial acetic acid in 100% ethanol)</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>GIBCO</td>
			<td>-</td>
			<td>monodispersion mixture</td>
		</tr>
		<tr>
			<td>Heating pads</td>
			<td>-</td>
			<td>-</td>
			<td>follow institutional protocol</td>
		</tr>
		<tr>
			<td>Ice buckets (x2)</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator with orbital rotation</td>
			<td>-</td>
			<td>-</td>
			<td>must be capable of maintaining 37&#176;C, shaking at 220-225 RPM (for collagenase digestion of mammary tissue)</td>
		</tr>
		<tr>
			<td>Isoflurane anesthesia</td>
			<td>-</td>
			<td>-</td>
			<td>follow institutional protocol</td>
		</tr>
		<tr>
			<td>Light microscope or digital camera</td>
			<td>-</td>
			<td>-</td>
			<td>visualizing whole mounted mammary epithelium and/or acquiring images</td>
		</tr>
		<tr>
			<td>Mechanical homogenizer</td>
			<td>Fisher Scientific</td>
			<td>-</td>
			<td>TissueMiser or alternative models</td>
		</tr>
		<tr>
			<td>Mineral oil, pure</td>
			<td>Sigma-Aldrich/ ACROS Organics</td>
			<td>8042-47-5</td>
			<td>long-term storage of cleared mammary gland whole mounts</td>
		</tr>
		<tr>
			<td>Oxygen tanks for anesthesia vaporizer</td>
			<td>-</td>
			<td>-</td>
			<td>follow institutional protocol</td>
		</tr>
		<tr>
			<td>Paper towels or delicate task wipes</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Positively-charged microscope slides</td>
			<td>Thermo Scientific</td>
			<td>P4981-001</td>
			<td>mammary gland tissue whole mounts</td>
		</tr>
		<tr>
			<td>Postoperative analgesic</td>
			<td>-</td>
			<td>-</td>
			<td>Institutional protocol</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td></td>
			<td></td>
			<td>body weight measurements of animals, proper dosing of pain medication</td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>-</td>
			<td>-</td>
			<td>electric clippers, or other</td>
		</tr>
		<tr>
			<td>Staining jars</td>
			<td>-</td>
			<td>-</td>
			<td>minimum of 1 per chemical wash, size appropriate for the number of slides, glass preferred</td>
		</tr>
		<tr>
			<td>Sterile field drapes</td>
			<td>IMCO</td>
			<td>4410-IMC</td>
			<td>used during transplantation</td>
		</tr>
		<tr>
			<td>Sterile scissors and forceps x3 (autoclaved)</td>
			<td>-</td>
			<td>-</td>
			<td>autoclave surgical tools used for donors and recipients</td>
		</tr>
		<tr>
			<td>Syringes: 5 mL (or greater)</td>
			<td>-</td>
			<td>-</td>
			<td>for sterile filtration of collagenase digestion media</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Worthington</td>
			<td></td>
			<td>monodispersion mixture</td>
		</tr>
		<tr>
			<td>Waste collection receptacle for liquids (poured or aspirated)</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Wound clip applier, clips, and removal tool</td>
			<td>Fine Science Tools</td>
			<td>12020-00</td>
			<td>Closing the skin incision over the interscapular white pad pad</td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Fisher Scientific</td>
			<td>X3S-4</td>
			<td>clearing mammary gland whole mount slides after staining</td>
		</tr>
	</tbody>
</table>

rat mammary epithelial cell transplantation into the interscapular white fat pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

Donor and recipient mammary glands 
The steps to isolate and prepare rat mammary epithelial cells for transplantation are shown in Figure 1A. At 4 weeks of age, the endogenous mammary gland of the donor rat has begun maturation and epithelium can be visualized on whole mounted slides stained with alum carmine (Figure 1B). One donor rat at this ag...

Watch this Scientific Journal Video about Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad at JoVE.com

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

This protocol has been used for over 40 years and remains the only reliable method for mammary cell transplantation assays in the rat. This method is widely used to interrogate mammary cell autonomous versus host effects in breast cancer susceptibility research. By grafting in the interscapular fat pad, this method circumvents having to clear the endogenous membrane epithelium which is not effective in rats.The method is therefore less invasive and the endogenous mammary glands can be used as an internal control. To begin this procedure, place a euthanized female rat on its back and secure it with pins. Spray the entire ventral surface with 70%ethanol.Make a sagittal Y-shaped incision allowing access to thoracic, abdominal, and inguinal mammary glands. Use scissors to dissect all the mammary gland tissue from the donor rat and remove all visible lymph nodes. Next, use forceps to extract the mammary tissue.Place it in a labeled 60 millimeter dish containing 500 microliters of serum-free DMEM F12 media and keep it on ice. Then use scissors to finely mince the mammary tissue. First, carefully turn the body of the donor animal over to place it in a prone position and secure it with pins.Spray the head and upper back with 70%ethanol. Locate the base of the skull and make an incision beneath the occipital condyles, insert sharp scissors beneath the skin and cut the skin away from the skull including the sides of the head. Next, use bone cutters or strong scissors to cut the skull along the midline from the occipital to frontal bones while making sure to keep the blade as superficial as possible and angled upward to prevent the destruction of the underlying brain tissue.Use Rongeurs or strong forceps to peel away the bone. Insert the tool lateral to the cerebellum to break the bone on either side. Sever the connections to the meninges by fully exposing the auditory canal.Use curved fine-tipped forceps to lift the brain out of the skull and record its weight. Use a fresh start of supplies for additional donor brains. Immediately transfer it to a 15 milliliter tube containing an equal amount of media that is stored on ice.Homogenize the brain for 10 to 15 seconds on a low speed. Then filter the homogenate to remove large pieces. Store the liquid on ice until needed.First, cut one centimeter off the end of a 1, 000 microliter pipette tip and manually place it on the pipettor. Use a cut pipette tip to transfer the minced donor tissue from each 60 millimeter dish to the collagenase digestion tube. Gently mix the minced tissue by pipetting it up and down one to two times.Then secure the samples in a horizontal position in the shaking incubator. Allow the samples to digest for 1.5 to two hours at 37 degrees Celsius while shaking at a speed between 200 and 220 RPM. When the donor mammary gland tissue is fully digested, centrifuge the suspension at 1, 200 times g and at four degrees Celsius for 10 minutes.After ensuring a pellet has formed, use a pipette to remove the fat layer or carefully pour off the supernatant. Gently resuspend the pellet in 10 milliliters of fresh DMEM F12 media. Repeat this process to thoroughly wash the cells.Prepare one 50 milliliter tube with a 40 micrometer strainer for every donor animal. Pre-wet the strainer with at least one milliliter of DMEM F12 media. Then use a pipette to pass the cell suspension through the filter to collect ductal fragments or organoids.For organoids, keep only the cells that remain in the filter. Invert the cell strainer over a new 50 milliliter tube and rinse with any volume of sterile media required to collect all the cells. After this, pellet the cells once more and resuspend in a small volume of media for counting.Prepare single batches of donor material for all the transplant recipients by combining equal volumes of the cell suspension with 50%brain homogenate for every site of transplantation. First, identify the base of the skull and start of the vertebral column on a recipient animal. Approximately one-third of the way down the spine, shave a three centimeter by two centimeter area on the upper thoracic portion of the back.Next, prepare the sterile field that will be used for surgery and arrange all needed supplies. Flush the Hamilton syringes with sterile DMEM F12 media to prevent the loss of cells. Load the entire volume of donor material into a separate syringe for each condition.After the syringe is fully loaded, invert it and press the plunger slightly to remove air bubbles at the tip of the needle. Keep the syringe on ice throughout the procedure. Ensure the animal remains unresponsive to deep stimuli with a firm toe pinch.Then use a sharp surgical blade to make a small interscapular incision. Locate the medial blood vessel for orientation and use forceps to lift the skin on one side of the incision. Hold the skin away from the fat pad and avoid the underlying brown fat while the transplant is performed.Next, insert the needle into the graft site. Carefully inject 40 microliters of the cell mixture into the interscapular white fat pad tissue. Remove the needle slowly.Hold the tissue in place and allow the transplanted cells to settle for three to five seconds. Repeat the entire injection procedure as previously described at the second site of transplantation. After this, close the surgical wound using wound clips or sutures and then discontinue the anesthesia.After sacrificing the animal, prepare it for dissection. Identify the medial blood vessel that separates the graft sites in the interscapular fat pad. Excise the entire pad as a single piece of tissue.Place the tissue on a positively charged microscope slide for whole mount. Place the slides in 70%ethanol for seven to 10 days to de-fat the tissue. Then prepare the alum carmine stain and process the slides when the tissue is sufficiently opaque.When processing is complete and the tissue on the slide has cleared, use low powered light microscopy or high resolution digital photography to acquire images of the slides for analysis. In this study, mammary epithelial cells are transplanted using a technique optimized for working with rats where the isolated mammary epithelial organoids from donor rats are grafted into the interscapular white fat pad of recipient rats. The presence, absence, or abundance of epithelium can be evaluated to determine success of the experiment as well as the autonomy of effects related to experimental variables.A reciprocal transplantation experiment where each factor has two levels such as wild type versus knockout will create four transplant groups for hypothesis testing. The representative whole mounted interscapular fat pad shown here contains positive epithelial outgrowth at both sites of transplantation. When analyzing the slides, lack of epithelium in the interscapular fat pad across many samples may indicate a technical problem with the procedure and should be treated as a negative outcome.Donor epithelial outgrowth can also be compared to the endogenous mammary gland and may facilitate additional conclusions. In the example shown here, the results of reciprocal transplantation using wild type and CDKN1B knockout rats suggested non-mammary cell autonomous effects on mammary gland development. Remember donor epithelial cells and organoids must be prepared carefully and kept alive until injected into the recipient animal.It is imperative to plan ahead and to perform the procedure carefully and without any interruptions. Primary carcinogenesis assays can be performed following transplantation to interrogate mammary cell autonomy in the etiology or treatment of breast cancer. Gene editing can also be incorporated to genetically engineer the donor cells or the recipient rat.As the sole method of performing mammary epithelial cell transplantation in rats, this procedure facilitates mammary gland development as well as carcinogenesis studies. This technique is particularly important for researching interactions between breast cancer cells and/or mammary epithelial cells with the host microenvironment. It enables studies in mammary gland development as well as breast cancer research.Those attempting this technique for the first time should optimize donor cell preparations to ensure maximal cell yield and viability, then perform small pilot experiments to practice surgical procedures and determine cell numbers to inject and also determine post-surgical incubation times required for experiments.

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