Transgenic and gene knock-out techniques and burn research

Journal of Surgical Research 123, 328 –339 (2005) doi:10.1016/j.jss.2004.06.001

RESEARCH REVIEW Transgenic and Gene Knock-Out Techniques and Burn Research Steven E. Wolf, M.D.,*,1 and Kenneth J. Woodside, M.D.† *Department of Surgery, University of Texas Health Science Center—San Antonio, San Antonio, Texas; and †Department of Surgery, University of Texas Medical Branch, Galveston, Texas Submitted for publication January 12, 2004

The development of transgenic technology has given researchers a powerful tool to examine biological effects, and the response to injury is no exception. Techniques such as pronuclear injection, targeted homologous recombination, and Cre/loxP gene excision are being used to construct animals with specific genetic designs; these are exploited to learn the role of genes in the response to severe burn. We review the construction of transgenic animals, pitfalls and benefits of this relatively new technique, and how this technique has been used in burn research. © 2004 Elsevier Inc. All rights reserved.

Key Words: transgenic; knock-out; burn. INTRODUCTION

Great strides have been made in burn research through in vitro, animal, and clinical research using all research techniques available in biology. During conduct of this research, conclusions are made about severe burn and its treatment after completion of experiments designed in response to observations made from the natural history of the injury. These experiments are designed under the assumption that when a factor is present in a given condition, a certain effect happens. When the environment is controlled such that only a certain factor is added and everything else is the same, any significant effect is then associated with the factor. If the outcome effect disappears when the factor is not present or instead removed while everything else

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To whom correspondence and reprint requests should be addressed at U.S. Army Institute of Surgical Research, 3400 Rawley E. Chambers Avenue, Building 3611, Fort Sam Houston, TX 782346315. E-mail: [email protected].

0022-4804/05 $30.00 © 2004 Elsevier Inc. All rights reserved.

is controlled, then a causative relationship is established. This is the classical modification to Koch’s postulates. An example of this principle in burn research is the observation of septic shock (effect) in patients with high numbers of bacteria in unattended wounds (factor). In fact, when this is recapitulated in controlled experiments whereby anesthetized animals undergo a severe burn and inoculation with high numbers of virulent organisms while other similarly treated animals are not inoculated, septic shock and its ramifications are found in the inoculated animals, thus demonstrating a clear association of high numbers of organisms in a burn wound and septic shock [1]. When these organisms are controlled with debridement or topical anti-microbials such that high numbers of organisms are no longer present, thus removing the factor, the ramifications of septic shock (effect) disappear [2, 3]. This relationship firmly links high numbers of virulent organisms in a burn wound with septic shock in the severely burned by a cause-and-effect relationship. This simple example is given to remind us how biological research is done at its foundation, and in fact, all new techne and means to control experiments must adhere to these principles as new ways of basic scientific investigation. In the last decade, cell biology and gene technology have advanced to the point where genetic manipulation of whole organisms can be accomplished, thus providing means for specific control of gene and protein expression within an experimental environment. This highly technical, complex, and elegant means of experimental control by selectively introducing (or deleting) genes in an animal through exogenous DNA or inducing a specific mutation, then placing it in an experimental condition to see the effect is collectively re-

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FIG. 1. Pronuclear injection. A single cell egg (E) is held at the end of a holding pipette (HP) with suction. A finely drawn injection pipette (IP) is used to introduce 1 to 2 pL of DNA solution containing the transgene into the pronucleus (pn) of the egg. The egg is approximately 100 ␮m in diameter. (With permission from Brenin DR, Talamonti MS, Iannaccone PM, Surg. Oncol. 6:99 –110, 1997, Elsevier Science Ltd.)

ferred to as transgenic research. You might find this research technology under other descriptors, such as “knock-out,” “knock-in,” “null,” “conditionally expressed,” or “genetically over-expressed,” depending on the genetic design of the animal. However, these experiments are nothing more than a means to an end, i.e., an elegant way of controlling an experimental condition so that scientific conclusions can be made about certain biological events. Burn research has taken advantage of transgenic animals in the search for mechanisms of responses to burn and how these mechanisms might be exploited to patient advantage. In this paper, we will briefly review the methods of creating transgenic animals, potential advantages and pitfalls, then review the burn literature for those experiments using burned transgenic animals to shed light on the effects of severe burn and burn treatment. FORMATION OF TRANSGENIC ANIMALS

Transgenic technology involves the ability to introduce exogenous genetic constructs (i.e., transgenes) into the genome of a whole animal in such a way that it is stably passed to its progeny [4]. The methods required to insert the genes into the germ line are complex, but once established, it is simple to maintain the line through breeding. Several methods are currently used to establish the genomic modification. We must admit that we are only bystanders to this process with no personal experience, aping what others have written in these regards.

Pronuclear Injection

Pronuclear injection of single-cell embryos is a common approach to generation of transgenic animals [5, 6]. Fertilized ova are obtained and injected with 1 to 2 pL of solution containing several copies of a transgene construct using an ultrathin pipette (Fig. 1). The transgene typically consists of a DNA sequence with a general promoter to drive expression as well as tissuespecific promotors to target expression in specific cell types. After one cell division cycle, the resulting embryo is placed into the oviduct of a pseudopregnant female. If successful, the transgene will integrate into the genome, typically with several copies of the transgene integrated end-to-end in tandem at the same place in the genome (concatemeric insertion). At birth, 5 to 40% of the resulting animals will be transgenic. A problem with this technique is the random nature of gene insertion, referred to as position effect. The DNA can enter the genome at regulatory elements for other genes causing an increase or decrease in its expression to environmental conditions that may or may not be desirable or predictable. Insertion of the gene may also disrupt native genes important in development and normal responses of the animal. This feature makes results from experiments difficult to interpret unless the site of insertion and its regulation are clearly known. Position effect can also interfere with reproduction, preventing establishment of a transgenic strain. Another problem with this technique is that the copy number of transgenes cannot be controlled, and thus

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technique: oncoretroviral vectors and lentivirus vectors. Oncoretroviral vectors integrate easily into the genome, usually as a single copy into multiple loci of the host genome [9], in opposition to pronuclear injection that inserts the transgene into a single site with many copies. Thus, the problem of position effect and copy number remains, or may even be more pronounced. An additional problem with oncoretroviral vectors is the common occurrence of gene silencing through trans-acting factors that bind to viral promotors, or DNA methylation. Thus, the transgene insertion is not stable. This disadvantage can be over-come by using lentivirus vectors, which are not as subject to silencing [10]. Another disadvantage is the limitation on transgene size (8 kilobases) that can be assembled via a virus particle. Embryonic Stem Cells

FIG. 2. Depiction of viral infection of cells in an early embryo. Some of the cells will be infected with the transgene and enter into the genome, including some germ line cells. When the mature animal is mated with a wild-type animal, a portion of the offspring will be heterozygous for the transgene. Heterozygous littermates can be mated to produce animals homozygous for the transgene.

the expression of the transgene is unpredictable. The copy number is of course correlated with level of expression, but expression is generally affected more by local factors related to insertion site than by copy number [7]. Lastly, transgene expression is sometimes silenced by methylation or chromatin rearrangement, which progresses with age of the animal and perhaps with successive breeding of the transgenic line [8]. Retroviral Transfer

Another method of producing transgenic animals is to infect early stage embryos with retroviral constructs. Co-culture with these viral particles leads to stable insertion of the transgene into the genome of a fraction of the cells of the embryo. Thus, the animal will be a chimera at birth with a proportion of the cells containing DNA from the infected stem cell and the rest from the donor embryo. Functional sperm or ova from the animal may then contain the stem cell genome. If the animal is mated, a portion of the offspring will then have all its cells with the transgenic genome. These offspring can be mated with one another to produce a line of homozygous transgenic animals (Fig. 2). Benefits of this technique over pronuclear injection lie in its technical simplicity. Expensive equipment such as a micromanipulators and visualization of the pronucleus are not required for efficient transfection. Two types of retroviruses are currently used in this

Embryonic stem cell transfection and embryo injection is the method of choice for a powerful application of transgenic technology, targeted gene knock-out by homologous recombination [11]. Embryonic stem cells are obtained from the inner cell mass of the blastocyst [12] through repeated disruption of the blastocyst after adherence to a special substratum. After 28 days, stem cells are selected and expanded. Stem cell pluripotency and stability of a diploid karyotype are ensured through markers [13], but the proof remains in the pudding; these cells must still be tested to ensure production of a stable chimera. The genome of these potential stem cells can be altered through injection or transfection with a constructed plasmid that integrates into the genome, then placed into [14] or on an embryo [15] for integration into the developing organism. The resulting offspring will be a chimera, and the homozygous transgenic animals can be isolated through selective breeding. The use of stable stem cell lines for generation of transgenic animals has a unique advantage in that rare nuclear events such as targeted homologous recombination can be exploited to provide “custom” modifications of the genome through targeted mutagenesis. Literally, millions of stem cells can be treated with a transgene in a targeting vector that contains flanking sequences homologous to those flanking the gene of interest. Because of the specificity of these flanking regions, the targeting vector is “traded” for the gene of interest with its flanking regions during homologous recombination—a rare nuclear event (Fig. 3). This inserted transgene will generally include a selectable marker, such as neomycin resistance gene, which is substituted for the functional gene. With successful recombination, cells with replacement of the functional gene by a neomycin resistance gene are selected by culture in neomycin. Often, targeting vectors will also be constructed with other transgenes that allow selection against recombination at incorrect locations

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FIG. 3. Homologous recombination. A plasmid designed to include a flanking sequences (A, C) to a gene of interest (B) is transfected into stem cells. Because of the similar sequences, these flanking sequences can conjoin to the native DNA with a spontaneous exchange of gene B for a gene that contains a product that confers resistance to neomycin. Thus, gene B has been traded for a neomycin resistance gene. By deduction, gene B is no longer present in this cell and so has been knocked-out. This stem cell line can be selected by culture in neomycin. The selected transgenic cells can then be introduced into growing embryos for incorporation into a developing animal. Homozygotes for this genetic manipulation can then be chosen through breeding.

(non-homologous). These stem cells are now null or knocked-out for the gene of interest because this gene has been deleted in favor of the inserted transgene. This cell line can be expanded and introduced into an embryo as described above. Alternatively, the targeting DNA vector can be designed such that the inserted transgene is not an inactivating sequence, but instead increases the action of the promotors and decreases the action of stop sequences around the gene, thus causing increased expression in the grown animal. Confirmation of Stable Transgene Insertion

Because transfection with the gene of interest is not certain with any technique, a method to discern presence of the gene of interest in an animal is necessary. The simplest method is to design a transgene construct that contains a dominant gene for a distinctive coat color in addition to the gene of interest. At the birth of a litter, those successfully transfected can be differentiated by hair color [16]. The most commonly used method is verification of gene presence by polymerase chain reaction using highly specific primer sequences for the constructed transgene. DNA is isolated from tail biopsies and is analyzed by PCR for the presence of the gene or gene markers associated with the transgene. Southern blots can also be done after endonuclease digestion of DNA, but is more time consuming. Lastly, dot plots can be used for denatured bacterial or viral DNA applied to a solid membrane not normally found in the animal’s chromosome. Once the line is established, the transgenic animals can be used in experiments. A note should be added

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regarding proper controls for such experiments, which ideally are littermates, thus minimizing gene changes present with natural diversity. The parents would be heterozygous for the gene of interest, and the progeny then will be either wild-type, heterozygous, or homozygous. In practice, this can be difficult as the line should be maintained through several generations to ensure stability that is most easily done by pairing homozygotes. It is conceivable that once the line is established, it can be passed back through by pairing wild-type to homozygous, giving rise to some heterozygotes through which the proper controls can be obtained. BENEFITS AND PITFALLS OF TRANSGENIC RESEARCH Benefits

Probably the greatest benefit of using transgenic mice in burn research is that it has enabled precise research into mechanisms of genetic responses at the whole body level. The use of animals without a gene of interest (i.e., knock-out or null) enables deletion of a particular molecule from the response. A pertinent example is the use of tumor necrosis factor-␣ (TNF-␣) null mice to determine if lymphocyte apoptosis after burn is related to the presence of this molecule. This experiment is reasonable based on the observation that levels of TNF-␣ are increased after burn [17], as is lymphocyte apoptosis [18]; thus the two are associated. What remains to establish cause and effect is the determination that if TNF-␣ is absent after burns through experimental means, then lymphocyte apoptosis should be also. Investigators showed that lymphocyte apoptosis levels in the thymus and spleen were maintained even in TNF-␣ null mice, eliminating TNF-␣ as the causative agent in this observation [19]. This experiment was elegant in that the investigators completely abolished TNF-␣ from the animal through genetic regulation, eliminating vagarities present with the use of inhibitors and such that are always questioned by complete penetrance of effect, dosing issues, etc. Another clear benefit of using transgenic animals is the ability to selectively increase expression (levels, preponderance, number) of a particular molecule to firmly establish association between its presence and an effect. An example of this would be experiments in mice that constitutively over-express cardiac-specific I␬B-␣. I␬B-␣ over-expression leads to inhibition of NF␬B translocation to the nucleus in response to an inflammatory signal that leads to transcription of a number of genes. Investigators showed that in mice over-expressing I␬B-␣ thus inhibiting NF␬B translocation, TNF-␣ secretion from cardiac myocytes was diminished after severe burn—which was associated with improved cardiac contractile function [20]. This experiment showed that a transgenic animal with in-

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FIG. 4. Cre recombinase excision. A gene with a tissue specific promotor, the gene for Cre recombinase, and loxP sites surrounding a stop sequence in front of a gene of interest. In the cells with expression of the tissue specific promotor, Cre is expressed. This enzyme then recognizes the loxP sites, and excises the stop sequence in front of the gene of interest. This now allows free transcription of the gene of interest limited only to those cells with Cre expression under the control of a tissue specific promotor. (With permission from Hardouin SN, Andras N, Clin. Genet. 57:237–244, 2000, Munksgaard International Publishers Ltd.)

creased cardiac expression for a particular molecule (I␬B) inhibited NF-␬B translocation, which is required for increased TNF-␣ secretion by the heart cells after burn and for the development of cardiac dysfunction after injury. This technology can be further expanded by designing constructs that express genes at times specified by the investigator and is silent at other times. Two such approaches are currently used. The first involves a tetracycline inducible gene expression system, which is a transgenic system containing a DNA sequence for a transcriptional regulator AND the gene to be controlled. The transcriptional regulator is a hybrid of two genes. One of these represses tetracycline resistance originally found on an operon in Escherichia coli. The other is a trans-activation domain of the herpes virus protein VP-16, the protein product of which promotes expression of genes found after a specific hybrid promotor. Activity of the VP-16 regulator is controlled by the presence or absence of tetracycline, which is at the whim of the investigator. In the presence of tetracycline, the transcriptional regulator is not functional, thus the gene of interest that follows the specific hybrid promotor in the transgene is not transcribed. In the absence of tetracycline, the gene is transcribed [21]. A second approach is to exploit recombinase enzymes found in bacteriophage. The one most often used is the Cre recombinase, which excises DNA between two 34 base pair consensus sequences (loxP sites) including one

of the loxP sites, then splices the ends together. The probability that a wild-type genome contains such sequences is extremely low, thus a system that employs Cre to excise loxP sites can be assumed to function only on those introduced by transgenic technology. A transgene can be synthesized that contains 1) the desired gene to insert into the genome with its flanking segments for homologous recombination, 2) a gene for selection of recombined cells such as neomycin resistance gene, 3) the gene for Cre under the control of regulatory elements for tissue specificity, and 4) deliberately placed loxP sites surrounding the introduced DNA sequence that the investigator wishes to excise in specified tissues based on the above tissue specific regulatory elements. In the designated tissues that express Cre, the DNA between the loxP sites is excised. This excised area might include the neomycin resistance gene (which can interfere with normal expression of surrounding genes), a stop sequence that inhibits transcription in every tissue except that which allows Cre transcription (Fig. 4), or a gene that the investigator wishes to selectively knock-out after it has been eliminated from a stem cell line by standard methods, then replaced (“knocked back in”) in that stem cell line and selectively knocked-out in specific tissues expressing Cre in the resulting animal. An animal that contains the Cre/loxP system is said to be “floxed,” and has genes modified only in certain tissues. An additional level of control can be obtained by fusing the Cre gene with a mutant estrogen-binding domain. In the presence of estrogen, gene expression proceeds, but can be inhibited in the presence of an estrogen antagonist such as tamoxifen. These discussed techniques indicate that very complex systems can be constructed to not only increase or decrease expression of a gene and its product in the whole animal, but also to direct the change in genome to one cell type (by exploiting regulatory elements specific to that cell type), leaving the rest of the organism alone. Therefore, changes in one cell type can be examined, including effects of changed expression in one cell type on other non-affected cell types. These changes in expression can be further exacted to specific times to change gene expression either before or during an experiment. This in particular is attractive in that it allows normal development outside of experimental conditions. It should be clear, then, that these techniques are theoretically very powerful in detecting the relevance of gene expression changes in physiological effects. However, these techniques are not without problems. Some of the pitfalls to transgenic research have already been mentioned, among them position effect and copy number for injection and viral techniques. The targeted approaches avoid these, but have their own problems as well related to the complexity of the technique. Another general “problem” is the specificity of

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the model, in that only a single gene of interest is modified. In trauma and burn research, the response to injury appears to be redundant, with the overall response the result of changes in many molecules—many of which have very similar functions. Therefore, inhibiting one response at the gene level may not have a significant effect, as other similar molecules are able to compensate. Furthermore, conclusions from any changes that are detected in an experiment with transgenic animals must be made carefully, in that the system is fundamentally changed from conception with continual absence (or presence) of a factor that is almost certain to continuously affect many surrounding molecules (except in the very complex systems described above). In fact, the intracellular (or extracellular) environment can be seen as a matrix of molecules, and removing or adding a factor may or may not have linear effects on its surrounding partner, but in addition could have distant non-linear effects through a series of unidentified pathways that on the surface do not appear to be significant (i.e., Chaos Theory). Another significant pitfall in transgenic research is related to the phenotypic response to the gene alteration. A DNA sequence of interest is changed, but yet the cell is functional in normal respects and can be used to form a whole animal. Oftentimes, these animals are indistinguishable phenotypically from their wild-type littermates, with normal development. This finding then calls into question the over all relevance of a specific gene change if normal development of the animal ensues. On the other hand, it could be surmised that a particular gene was conserved and is useful (and necessary) in the response to injury, and does not need to have any other effects. Also, as development is an integrated process, altering a single gene may have effects on other systems that may result in the adult animal having a different physiology than if the gene just disappeared in adulthood. Transgenic research is limited somewhat by the availability of animal models. The mouse is the most ideally suited mammal at present for this type of research. Reasons are that it is small with a relatively rapid breeding cycle so that homozygous transgenic stock can be reached in a timely fashion. Also, the mouse genome is known, which allows for easy gene identification and knowledge of surrounding promotors/inhibitors and such on the DNA. Evidence for this is the ready commercial availability of over 3000 strains of transgenic mice. Recently, more and more transgenic rats have become available to researchers for similar research, as well as some other animals such as rabbits [22], sheep [23], and horses [24]. These particular animal models are sufficiently distant from humans to provide some comfort. However, as transgenic research in whole animals approaches the primate realm, expect ethical discussions

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and dilemmas to rapidly arise. Nonetheless, the relevance of research in non-human mammals—with different DNA than ours—must always be understood with that caveat. Finally, the last pitfall is more a personal observation on scientific communication. Because of the complexity of the model development with transgenic technology, it is often difficult to understand simply what the investigators have done. Sometimes two or three genetic modifications are made, requiring counterintuitive logic to discern exactly what has happened and what relevance it has to non-genetically modified systems. This complexity itself should not be seen necessarily as a pitfall, but is mentioned only to remind investigators and readers that communication of this type of research is often confusing, and writers should try to make the diction as simple as possible without unnecessary abbreviations or other assumptions of understanding. The discussion should always include a brief description of the model for the general scientific reader. TRANSGENIC RESEARCH IN BURNS

Thus far, transgenic animals are used in burn research, but relatively sparingly. On searching the Medline database using the terms transgenic and burns, 14 total articles returned compared to 40,279 for transgenic alone. Of these 14, only 8 were relevant to burn research. Using the terms knockout and burns, 22 articles were found, with only 14 related to burn research, compared to 25,529 articles using just the term knockout. Further digging with other terms revealed the use of transgenic animals in burn-related research primarily in three areas: cardiac function after burn, immune activity after burn, and in wound healing. Most of these are reviewed herein. Two interesting articles on the use of transgenic bacteria in burn wounds are also mentioned. Cardiac Function

The group at Parkland Hospital in Dallas has been active in using transgenic animals to examine heart function after severe burn. To develop a murine burn model of heart function, they performed an experiment demonstrating a 40% total body surface area burn in mice impaired cardiac contraction and relaxation. Burns also promoted cardiomyocyte secretion of TNF-␣, IL-1␤, and IL-6, and increased cardiomyocyte concentrations of calcium [25]. This background work lead to three studies using transgenic mice— one of which has been mentioned previously. They used a mouse over expressing I␬B to inhibit NF-␬B nuclear translocation in cardiomyocytes. They found that this experimental maneuver prevented TNF-␣ secretion and ablated cardiac contractile dysfunction after burn

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FIG. 5. Left ventricular (LV) function was examined after burn trauma in groups of I␬B over expressing mice and wild type littermates. Hearts were harvested 24 h after burn and perfused in vitro with LV function examined while coronary flow was increased. All values are mean ⫾ SEM, * ⫽ P ⬍ 0.05. (With permission from Carlson DL, White DJ, Maass DL, et al., Am. J. Physiol. Heart. Circ. Physiol. 284:H804 –H814, 2003, American Physiological Society.)

(Fig. 5) [20]. In another experiment, they showed that mice deficient for IL-1 receptor associated kinase-1 (IRAK-1) and thus attenuated IL-1␤ signaling also resisted LPS induced cardiac contractile dysfunction, implicating IL-1 in the post-burn process of cardiac dysfunction [26]. Lastly, they used mice deficient for the inducible form of nitric oxide synthase (iNOS) to show that cardiac deficits related to burn are diminished in the absence of iNOS, which was confirmed with pharmacological blockade of iNOS activity [27]. These experiments with transgenic mice have shed further light on cardiac contraction problems that develop shortly after burn. Immune System

Three groups are using transgenic mice for investigations into the immune system after severe burn. The first mentioned here, the group at Brigham and Women’s in Boston used a transgenic mouse with a specific MHC class II restricted T-cell receptor (DO-11.10) that recognizes only a 323–339 amino acid sequence of ovalbumin (OVA) not normally found in the mouse environment. This system can be exploited in interesting ways. First, the transgenic mouse can undergo severe burn, then the activity of the T-cells in the burned mouse environment can be specifically measured by exposing the animal to the specific antigen and observing T-cell responses. Additionally, these

mice can be immunized to the antigen before burn, then exposed again after burn to examine the response of a “mature” immune system. In their first experiment, they found that presentation of the OVA antigen after a severe burn stimulated proliferation of T-cells similar to antigen stimulated T-cell proliferation from transgenic mice that were not burned (Fig. 6). These T-cells had increased production of interferon-␥, intimating that burn primed T-cells for a Th1 type response as opposed to a Th2 response. This was in contrast to findings in wild-type mice that have Th2 type response to general antigen stimulation after burn. Interestingly, if the transgenic mice were immunized with the OVA antigen, then received a burn, mortality was significantly increased [28]. Increased mortality seen with the immunized transgenic mice, then, must be related to active and rapid increase in primed cell-mediated immunity. Alternatively, this may be a function of this particular transgenic mouse that may have the gene inserted into a critical region (position effect) or some other vagarity related to its altered genome that does not necessarily correlate activity of mature immunized cell-mediated immune processes with increased mortality. In a twist on this model, and to partially answer some of the above concerns, the investigators took CD4⫹ T-cells from these transgenic mice and adoptively transferred them to wild-type mice. These mice

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FIG. 6. Burn does not impair antigen-induced proliferation of lymph-node derived naïve CD4 T-cells. Male AND or DO-11.10 TCR transgenic mice were burned. Seven days later, lymph nodes were prepared for stimulation with specific antigens. The cultures were pulsed with 3H thymidine for 18 h of a 3-day culture period. Results shown as counts per min ⫾ SEM. (With permission from Kavanagh EG, Kelly JL, Lyons A, et al., Surgery 124:269 –277, 1998, Elsevier Science Ltd.)

were then burned to examine T-cell function in a nontransgenic environment. The T-cell responses can be tracked using a monoclonal antibody specific for the DO-11.10 T-cell receptor. In this experiment, they used wild-type mice with transgenic T-cells making up a population of the total T-cells. These mice were exposed to OVA while being burned. They found that burn did not affect the proliferative response to antigen in the first 3 days after injury compared to controls, indicating that burn did not affect the early response to antigen immunization. When the phenotype of T-cells was examined by culturing lymphocytes from burned and non-burned wild-type and transgenic mice with OVA and measuring the cytokine response, they found that Th1 type cytokines (IL-2 and interferon-␥) were suppressed 3 days after injury while Th2 type cytokines (IL-4 and IL-10) were not. Splenocytes had a slightly different response in that Th1 cytokines were not affected while a Th2 cytokine was significantly higher. These changes were even more evident 7 days after injury. So, it appears that early T-cell responses to antigen are conserved early after burn followed by a phenotypic switch from a Th1 type response to a Th2 response [29]. This has also been intimated in patients [30]. Further findings from this study with cultured lymphocytes without immunization revealed similar Th1 type responses in response to OVA stimulation from cells collected either 1 day or 7 days after injury, suggesting that the response of T-cells to a naïve antigen is similar regardless of time from injury [29]. The findings above suggest that with time, these early Th1 responses shift to a Th2 type response with time exposed to the antigen, consistent with what is observed clinically. The conclusions that can be drawn from this line of research is that injury and exposure to antigen induces a Th1 T-cell response similar to that seen in the non-injured. This response, however, does seem to be related to higher mortality, as seen in one of the

described studies as well as another that used a burn and staphylococcal superantigen exposure as stimulus [31]. These conclusions could not have been easily reached without transgenic technology. These investigators have also used other transgenic mice to examine the immune system after burn. Based on the supposition that IL-10 induction (a Th2 type cytokine) is protective after injury, they burned IL-10 knockout mice and compared their responses to wildtype controls. They found that survival was equivalent between groups, and Th1 type reactions still occurred early after burn without undue consequences [32]. This suggests that a key component of the Th2 type response (IL-10 expression) that occurs later after injury is not required for survival in this animal study. In another study, they examined the effect of burn on recombination activating gene (RAG) knockout mice that do not have a functional adaptive (cell-mediated) immune system, but do have a functional innate immune system (neutrophils, macrophages, etc.). They examined mortality compared to wild-type controls and found no differences. In addition, they did not find any differences in mortality between groups when cecal ligation and puncture was added 3 days after burn [33]. They performed other experiments where wildtype splenocytes were adoptively transferred followed by cecal ligation and puncture alone, and this was found to provide protection. It must be stated that these latter animals were not burned, and thus no conclusions can be made regarding this treatment after severe burn. Instead, we must return to the original data showing that absence of the adaptive immune system through transgenic means had no effect on mortality after burn and/or cecal ligation. This is of course of interest and should be examined in other models. Another study from the group at the University of North Carolina used transgenic female mice with a

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specific T-cell receptor for HY antigen (normally found in males). These mice then are similar to the transgenic mouse used in Boston with a T-cell receptor for OVA. They did an experiment where the mice were burned, and after 72 h splenocytes were obtained and stimulated with the HY peptide. In this experiment, burn decreased the splenocyte proliferative response to the antigen when compared to sham control. When they examined CD8 cells specifically, they found that intracellular IL-2 and interferon-␥ were increased, indicating a Th1 type response in these cells. CD8 cells are also called cytotoxic T-lymphocytes (CTLs), but in this study were found to produce cytokines more typical of T-helper cells (CD4 cells) [34]. Other groups have been active with the use of transgenic mice in examining the immune response to burn. A group at the University of Florida noticed that 3 h after burn, lymphocyte apoptosis and caspase-3 activity were increased in the thymus and spleen of burned mice, which was associated with increased Fas ligand expression. These findings were also present in tumor necrosis factor-␣ (TNF-␣) knock-out mice, indicating the effect was independent of TNF-␣ activity [19]. Apoptotic regulation of intestinal lymphoid populations has also been investigated using transgenic models. The University of Florida-Gainesville group demonstrated increased apoptosis of intraepithelial lymphocytes and lymphocytes in Peyer’s patches within 6 h of a 20% scald burn injury in mice. Furthermore, they determined that increased B cell and CD8⫹ T cell apoptosis was occurring in the burned mice and that this apoptosis was partially abrogated by the steroid antagonist mifepristone. FasL deficient mice demonstrated unchanged CD8⫹ T lymphocyte apoptosis after burn and did not demonstrate increased B cell apoptosis after burn, suggesting a differential apoptotic role in lymphocyte subsets [35]. In addition to the investigations of FasL and TNF-␣, the related surface protein Fas has also been investigating using a knockout burn model. Fas-deficient lpr (⫺/⫺) mice underwent burn and superantigen challenge. Lpr (⫺/⫺) mice had increased survival after burn, but splenocytes isolated from these animals expressed less TNF-␣, IFN-␥, and interleukin-2, suggesting that the death receptor Fas is integral to the inflammatory response post-burn [36]. From this data, it is intriguing to speculate that apoptosis after global activation may not always be beneficial after burn, despite the demands of such an expanded lymphocyte population. Recently, T cell receptor (TCR)-␦ (⫺/⫺) mice have been used to analyze the post-burn immunological response. A group at Brown University has investigated cross-talk between macrophages and ␥␦ T cells, which predominantly distribute in the intestine and skin, using this ␥␦ T cell-deficient model. Mortality was in-

creased and splenic macrophage production of the proinflammatory cytokines TNF-␣ and interleukin-6 was reduced in ␥␦ T cell-deficient mice after burn, suggesting a priming role for ␥␦ T cells [37]. Building on this concept, we investigated the role of ␥␦ T lymphocytes in burn injury-induced mucosal turnover. We found that gut epithelial apoptosis and proliferation in both wild type and TCR-␦ (⫺/⫺) mice were increased after burn, but TCR-␦ (⫺/⫺) mice had a lower level of apoptosis and proliferation compared to wild type mice— indicating that they had a lower rate of epithelial turnover [38]. Therefore, ␥␦ T cells are associated with increased TNF-␣ expression and gut epithelium turnover in the small bowel after severe burn. However, absence of ␥␦ T cells does not inhibit mucosal atrophy after severe burn, suggesting that gut mucosal atrophy after severe burn is a multi-factorial process associated with increased TNF-␣ activity, but that this activity is insufficient in and of itself to induce the pathologic response associated with burn. Recent studies have begun to investigate the interface between metabolism and immune function. Using an interferon (IFN)-␥ (⫺/⫺) mouse and 20% TBSA burn, the MGH group in Boston demonstrated that skeletal muscle hypercatabolism is IFN-␥-dependent and that lymphocyte proliferation post-burn is relatively normal in the IFN-␥-deficient animals [39]. Similarly, IL-6 has been demonstrated to be essential for hepatocyte glutamine transport after burn injury by using an IL-6 deficient mouse model [40]. Wound Healing

Another area of burn research where transgenic animals have been used is in wound healing. The group in Edmonton, Canada has developed a mouse that over expresses transforming growth factor-␤ (TGF-␤) under the control of a keratin-14 promotor (localized to the epidermis) to examine the effect of this peptide on wound healing after burn. They found that these mice had a significant reduction in the rate of reepithelialization after full-thickness skin excision, which was associated with increased levels of collagen in the wounds when compared to wild-type controls [41]. When this mouse underwent partial thickness burns with a CO 2 laser, it was found that TGF-␤ was significantly increased in the wounds of the transgenic mouse, and this increased expression was associated with slower wound epithelialization (Fig. 7). Expression of Type 1 collagen and hydroxyproline was again increased, similar to the findings with full-thickness skin excision [42]. These experiments indicate that increased TGF-␤ expression results in two undesirable effects: inhibition of wound epithelialization and increased collagen deposition—thus increasing scarring. This could not likely be shown effectively without transgenic techniques.

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Another group at Vanderbilt examined the effects of CXCR2, a chemokine that attracts neutrophils, on wound healing. In an experiment with transgenic mice without CXCR2 expression, they found that the transgenic mouse had significantly decreased neutrophil recruitment into a nitrogen mustard chemical burn that was associated with delayed wound healing [43]. Transgenic Bacteria

Investigators at the University of Arkansas performed an interesting study with transgenic bacteria that is very relevant to burn research. Infection with Pseudomonas is common after severe burn. During infection, the bacterium senses environmental changes and regulates the expression of its genes relevant for survival. The investigators isolated differentially expressed bacterial genes in burn wounds of mice, one of which was superoxide response regulator (soxR). Expression of soxR was found to be highly inducible with burn wound infection. The investigators then concocted a soxR knock-out Pseudomonas bacterium, which was found to be more sensitive to macrophagemediated killing, and exhibited a significant delay in causing systemic infections in burned mice [44]. So, these investigators showed an association between increased soxR expression in Pseudomonas bacterium and burn wound infection. When soxR expression was deleted in the bacteria, infection risk decreased, showing a clear cause-and-effect relationship between soxR and Pseudomonas burn wound infection in the classical sense. Similarly, the group at Stanford University demonstrated that Pseudomonas deficient in the polyphosphate kinase (PPK) gene is unable to form a thick biofilm. Furthermore, the bacteria is deficient in the quorum-sensing controlled virulence factors elastase and rahmnolipid, and has reduced ability to colonize burned mouse tissue [45]. Clearly, genomic modification of non-eukaryotic pathogens can also be a useful technology for burn research. CONCLUSIONS

The discovery of transgenic technology has vastly expanded the repertoire for investigators to examine physiology and the effect of injury. It provides for the design of very elegant models with numerous exciting possibilities for conclusions about the mechanisms of observed effects, and how our treatments might affect these effects and outcomes. However, this technology does have serious limitations that we hope we have enumerated. As far as burn research, the use of trans-

FIG. 7. Delayed wound healing in transgenic mice. Wound closure in wild type (WT), heterozygotes (HT), and homozygotes (HM) was assessed visually and histologically. Wounds were scored as open or closed. Forty-eight wounds were examined in each group

at 12 days and 16 days (* ⫽ P ⬍ 0.05, ** ⫽ P ⬍ 0.01). (With permission from Yang L, Chan T, Demare J, et al., Am. J. Pathol. 159:2147–2157, 2001, American Society for Investigative Pathology.)

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genic technology has been limited thus far. In particular, the immune response to injury seems to be quite suited for the use of transgenic animals, as well as wound healing. We look forward to such studies. Lastly, we all must remember that the use of transgenic animals for research is only a means to an end— namely, the definition of how things work and how this knowledge can be used to improve treatment of patients. Transgenic tools are a technology—we must still do the science. In fact, at times, it may be the wrong tool to use leading to inappropriate conclusions when a more conventional model would be more appropriate. Knowledge both of how these animals are constructed and of the potential shortcomings of this construction is important to consider when reading or conducting research with this technology. Alternatives to transgenic models such as the use of specific inactivating antibodies or, even more novel, the use of inactivating RNA sequences injected into wild-type animals, should also be considered to confirm findings from transgenic and gene knockout animal experiments. ACKNOWLEDGMENTS We wish to acknowledge the assistance of Eileen Figueroa and Steve Schuenke in the preparation of this manuscript. We also wish to acknowledge Dr. Paul Love, at the National Institute for Child Health and Human Development, who reviewed the manuscript for accuracy and relevance.

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