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Human Molecular Genetics Pages 2079-2088  

LCR-dependent gene expression in [beta]-globin YAC transgenics: detailed structural studies validate functional analysis even in the presence of fragmented YACs
Introduction
Results
   Pitfalls of structural analysis of [beta]-globin locus YAC transgenics
   Detailed structural analysis of the 248 [beta]-YAC transgenics reveals multiple rearrangements
   Retrofitting of a YAC with I-PpoI sites facilitates structural analyses
   Correct structural analyses of YACs allows investigation of structure-function relationships of the transgenes
   Variation in gene expression between littermates and between transgenic lines
Discussion
Materials And Methods
   PpoI retrofitting of the 155 kb [beta]-YAC
   YAC purification and microinjection
   Use of blood buffy coat-derived plugs for PFGE analysis
   Structural analysis of integrity of [beta]-YAC transgenics
   Copy number determination
   RNAse protection analysis
Acknowledgements
References

LCR-dependent gene expression in [beta]-globin YAC transgenics: detailed structural studies validate functional analysis even in the presence of fragmented YACs

LCR-dependent gene expression in [beta]-globin YAC transgenics: detailed structural studies validate functional analysis even in the presence of fragmented YACs

Kenneth R. Peterson1,2,*, Patrick A. Navas1, Qiliang Li1 and George Stamatoyannopoulos1,2

1Division of Medical Genetics, Department of Medicine and 2Department of Genetics, University of Washington, Seattle, WA 98195, USA

Received June 23, 1998; Revised and Accepted September 14, 1998

Yeast artificial chromosome (YAC) transgenesis is associated with a high frequency of deletions in the integrated transgenes. To determine the impact of these rearrangements on the ability to derive structure-function relationships using YACs, transgenic mice were generated with 248 or 155 kb [beta]-globin locus YACs. The transgenics were examined for structural integrity of the YAC using an approach of structural analysis that unambiguously demonstrates intactness of YAC transgene copies. Globin gene expression per copy of each integrated transgene and the profiles of globin gene expression during development were determined. Diverse deletion patterns were observed in one or more integrated YACs in all the 248 and most of the 155 kb transgenic lines we analyzed. However, when the structure of the major regulatory element of the [beta]-globin locus, the locus control region, was preserved, the genes of the [beta]-globin locus functioned normally and globin transgenes of both the 248 and 155 kb [beta]-YACs were expressed in a position-independent, copy number-dependent manner. Furthermore, the globin genes of both [beta]-YACs displayed normal developmental regulation. We conclude that YACs can be used for analysis of structure-function relationships of large genes or multigene loci in spite of the tendency for rearrangements and deletions of the integrated transgenes. However, detailed structural evidence for integrity and continuity of locus sequences is required for correct interpretation of functional data.

INTRODUCTION

Transgenic mice produced from large constructs including cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) have become useful tools for studies of gene regulation in the context of native loci and as mouse models of human disease (reviewed in refs 1,2). In contrast to simple recombinant constructs, YAC transgenes allow the inclusion of all gene sequences and cis-regulatory elements necessary for proper gene expression. In addition, the normal spatial arrangements are maintained between these various motifs. YACs, and more recently BACs (3), have an additional advantage over other transgenes in that mutations may be introduced by homologous recombination in the host organism without leaving exogenous DNA. Thus, the effect of a mutation may be studied throughout development, in an intact locus, in transgenic mice generated from these mutant DNA molecules. Examples of wild-type YAC transgenics that display proper expression patterns and levels are abundant (1,2) and reports of studies using mutant variants of these YACs are beginning to appear (4-8).

Perhaps the most utilized YAC transgenic system to date involves analyses using human [beta]-globin locus YACs ([beta]-YACs). Two such YACs were identified, one is 248 kb, the other is 150 kb (9), and both have been used in wild-type and mutated forms to generate transgenic mice (4-6,8,10,11). The wild-type versions show proper developmentally regulated globin gene expression (10,11) and mutant forms show altered expression phenotypes (4-6,8). Since our first report on [beta]-YAC mice (11), we discovered that structural analysis of YACs by conventional Southern blot hybridization and PCR could, in fact, fail to demonstrate integrity of YAC transgenes. This prompted us to develop new methodology for the study of YAC structure in mice (5,6). Identification of mice with intact YACs is important for correct interpretation of expression data, particularly in mice where mutant YAC constructs have been used. In this paper we report the detailed structural analysis of transgenic mouse lines carrying the 248 or a modified 150 kb [beta]-YAC. In addition, we demonstrate that the major regulatory element of the [beta]-globin locus, the locus control region (LCR), functions normally in both [beta]-YACs based on the correlation between copy number and level of globin gene expression and the small coefficient of variation in the per copy globin gene expression between lines.

Our data indicate that in spite of the presence of multiple integrations and deletions of YAC sequences, the function of the [beta]-globin locus remained normal, as long as the major regulatory element, the LCR, was intact. We conclude that regardless of the high frequency of deletions in the integrated YACs, YAC transgenics are very useful for functional analysis of large or multigene gene loci, provided that detailed structural analysis of the integrated loci is done. Such structural studies are especially important when the YAC transgenic mice display abnormal phenotypes.

RESULTS

Pitfalls of structural analysis of [beta]-globin locus YAC transgenics

In initial studies (11), the murine genomic DNA was analyzed by Southern blot hybridization of restriction digests and assayed by PCR for the presence of the arms of the YAC vector, which presumably represented the junctions of the [beta]-YAC construct and the murine DNA. The presence of [beta]-globin locus sequences was ascertained by hybridization to murine DNA with probes spanning ~60 kb of the locus (see Materials and Methods). These probes identified EcoRI fragments with sizes between 2.4 and 10.6 kb and were chosen in order to detect the presence of locus sequences from 20 kb 5[prime] of the [epsis]-globin gene to within the [beta]-globin gene of the [beta]-globin locus. When all diagnostic bands were present we concluded that the integrated YAC was intact (11). This approach was proven incorrect because, as we show below, the YACs integrated into the murine genome are usually fragmented. Positive signals by Southern hybridization indicating the presence of an intact YAC can be obtained when there is more than one fragmented YAC in the transgene genome and DNA fragments missing from one YAC copy are present in another integrated YAC. This approach also fails to detect the diversity of YAC integration and the fact that a mouse line can carry multiple copies of the YAC with different deletions.

Detailed structural analysis of the 248 [beta]-YAC transgenics reveals multiple rearrangements

Most of the [beta]-globin locus is encompassed on a 140 kb SfiI restriction enzyme fragment contained within the 248 kb [beta]-YAC. One SfiI site is between 5[prime]HS4 and 5[prime]HS3 of the LCR, the other is downstream of 3[prime]HS1 (Fig. 1). The goal of our structural analysis was first to detect, by pulsed-field gel electrophoresis (PFGE), the sizes of globin gene-containing SfiI fragments that existed in the genome of the transgenic mice and second, to resolve, in detail, the globin gene sequence contained in these fragments. SfiI fragments smaller or larger than 140 kb indicate the loss of at least one of the SfiI sites flanking the [beta]-globin genes. The size of the resultant fragment thus becomes dependent upon the juxtaposition of the nearest murine genomic SfiI site.


Figure 1. Diagram of the 248 kb [beta]-YAC. The 230 kb EcoRI human genomic fragment containing the [beta]-globin locus is shown as a line. YAC vector sequences and [beta]-like globin gene sequences ([epsis], G[gamma], A[gamma], [psi][beta], [delta] and [beta]) are shown as black boxes. The location of the LCR is noted; arrows above the line denote DNAse I-hypersensitive sites (HSs). SfiI restriction sites are displayed below the locus; the line above the locus delineates the 140 kb SfiI fragment used in assessment of [beta]-YAC transgene integrity. The locus spans 82 kb from 5[prime]HS5 to 3[prime]HS1; there is 39 kb 5[prime] flanking sequence and 109 kb 3[prime] flanking sequence. YAC components: TRP1 and LYS2, YAC selectable markers for tryptophan and lysine prototrophy, respectively; ARS1, yeast autonomous replicating sequence (origin of replication); CEN1, centromere; MMTneo, mammalian cell selectable marker for G418 resistance.

PFGE structures of the DNA isolated from [beta]-YAC transgenic mouse lines have shown that these mice usually contain multiple SfiI fragments of different sizes (Fig. 2). To define which globin genes were contained in each of these PFGE SfiI fragments, the approach (described in Materials and Methods) of hybridizing multiple PFGE lanes with separate probes spanning the [beta]-globin locus was applied (5,6,12).


Figure 2. Structure of [beta]-globin loci in 248 kb [beta]-YAC transgenic lines. Agarose plugs containing high-mass murine DNA were digested with SfiI and the DNA was fractionated by PFGE. DNA was transferred by capillary blotting and the membrane was cut into strips, each of which comprised one lane of the gel. Each strip was hybridized with a single probe; the entire probe set spanned the [beta]-globin locus. After washing, the strips were reassembled and autoradiography was performed. An image of the individual YAC copies and the extent of the sequence that each contained was thus obtained. The representation of the locus at the top shows the 140 kb SfiI fragment used to determine intactness of the locus. It spans from between 5[prime]HS4 and 5[prime]HS3 of the LCR to downstream of the HPFH6 breakpoint, thus encompassing most of the [beta]-globin locus. Probes utilized are shown as dotted lines below the locus map and are listed by Peterson et al. (5). Transgenic line numbers are indicated on the left, with the estimated sizes of individual YAC copies immediately adjacent to each line number. Individual YAC transgenes are represented as solid lines. The ends of the lines coincide with the last hybridization signal observed and do not necessarily indicate YAC copy sequence endpoints. 5[prime]HS4 and 5[prime]HS5 sequences were detected by standard Southern blot analysis (data not shown). The intensity of the bands was approximately equal to the band representing YAC copies that contained 5[prime]HS3, indicating a high probability of sequence continuity from 5[prime]HS5 through 5[prime]HS3.

Figure 2 graphically illustrates the structures of the YACs in each transgenic line, as determined by the pulsed-field gel analysis described in Materials and Methods. As shown, the nine transgenic lines contained from one (line 4) to six (line 10) SfiI fragments, most of which were fragmented. Most of the deletions occur at the 5[prime] or 3[prime] end, or both, of the YAC copies, indicating that loss of sequence was most likely a result of breakage during in vitro manipulation or microinjection. Apparent internal deletions or rearrangements were also observed, which may result from murine recombination processes in vivo. Alternatively, these sequences could also be individual 5[prime]/3[prime] deleted YAC copies of similar size that co-migrate to nearly the same position in the pulsed-field gel, but are not resolved by our PFGE conditions. It should be noted that in all lines containing [beta]-YAC copies spanning 5[prime]HS3 through A[gamma], 5[prime]HS5 and 5[prime]HS4 sequences were detected by Southern blot analysis (data not shown). The band intensities observed following hybridization with these two probes equaled the sum of the aforementioned intact YAC copies. These data indicate that the LCR is intact in these copies.

Some lines contain intact [beta]-globin loci and additional loci that have a deleted [beta]-globin gene (lines 1 and 3). The latter loci are expected to contribute to globin gene expression in the embryo and the fetus, but not in the adult. Thus, to properly analyze the function of the locus during development, the copies of the [epsis]- and [gamma]-globin genes of the [beta]-globin gene-deleted YACs must be taken into consideration when per copy [epsis]- and [gamma]-globin gene expression is calculated during the embryonic and fetal stages of development.

Several lines provide excellent examples of why detailed structural analyses such as those described in this paper are required to unambiguously interpret structure-function relationships of YAC transgenic mice. For line 6, if only standard Southern blots of restriction enzyme-digested murine DNA in the 500 bp to 23 kb size range were analyzed, we would conclude that these transgenic mice contain an intact [beta]-globin locus. In reality, this line has three YAC fragments, each of which contains only a portion of the complete locus. Line 7 has five YAC copies. However, four of these YAC fragments are missing 5[prime]HS3-5 of the LCR and the globin genes should be functionally silent. Only one contains a complete [beta]-globin locus with an intact LCR and therefore only this transgene should express normally. When globin gene expression is measured, only one copy should be used for calculation of per copy gene expression.

Retrofitting of a YAC with I-PpoI sites facilitates structural analyses

A 150 kb [beta]-YAC with 39 kb upstream of the LCR and 43 kb 3[prime] of the [beta]-globin gene has been identified (9). This YAC has an identical, or nearly identical, 5[prime] end to that of the 248 kb [beta]-YAC (Fig. 3A), but its 3[prime] extends 23 kb downstream of 3[prime]HS1, while the 248 kb [beta]-YAC extends 109 kb downstream of 3[prime]HS1.


Figure 3. Diagram of the 155 kb [beta]-YAC (Ppo-155 [beta]-YAC) and complete structural analysis of two Ppo-155 [beta]-YAC lines. (A) The 150 kb [beta]-YAC (A201F4; 9) was retrofitted to produce the Ppo-155 [beta]-YAC as described in Materials and Methods. The elements of the diagram are as described in the legend to Figure 1. The I-PpoI sites introduced at the insert-vector junctions by retrofitting are indicated below the locus. The 144 kb I-PpoI fragment used for structural analysis of YAC transgenics is shown above the locus drawing. This fragment encompasses the whole human genomic insert and is utilized in place of the 140 kb SfiI fragment used for 248 kb [beta]-YAC structural studies since the 3[prime] SfiI site is missing in the Ppo-155 [beta]-YAC. This YAC contains the 82 kb locus (5[prime]HS5 to 3[prime]HS1), 39 kb 5[prime] flanking sequence and 23 kb 3[prime] flanking sequence. (B) Below the locus map autoradiographs of YAC transgene structures for Ppo-155 [beta]-YAC lines 1 and 8 are displayed. Solid lines between the map and the autoradiographs indicate the probes utilized. The sizes of the YAC copies are indicated on the right and the location of [lambda] PFGE markers is shown on the left.

Because the SfiI site flanking 3[prime]HS1 is not present in the 150 kb [beta]-YAC, it is difficult to use this YAC for the type of structural analysis described in the previous section. Therefore, we introduced I-PpoI intron-encoded restriction enzyme sites at the junctions of the pYAC4 vector sequence and the cloned globin locus insert sequence using a series of two retrofitting vectors, pUC OK and pUC WAN (13). The structure of the resultant 155 kb [beta]-YAC is shown in Figure 3A. Digestion of agarose plugs containing YAC-bearing yeast DNA with I-PpoI removes the YAC vector arms and produces an ~144 kb fragment containing the entire [beta]-globin locus insert (Fig. 3B). The fragment of this so-called Ppo-155 [beta]-YAC is perceptibly smaller than the uncut YAC on pulsed-field gels.


Figure 4. Structure of [beta]-globin loci in Ppo-155 [beta]-YAC transgenic lines. Agarose plugs were digested with I-PpoI and the DNA was subjected to PFGE. The DNA was Southern blotted and the membrane was cut into strips representative of one lane of the gel. Each strip was hybridized with a radiolabeled probe, one to detect the 5[prime] end of the [beta]-globin locus, one to detect the middle and one to detect the 3[prime] end. After washing, the original membrane was reassembled and an autoradiograph produced. An image of each YAC copy within a line displaying sequence continuity and approximate deletion breakpoints was obtained. The representation of the locus at the top shows the 144 kb I-PpoI fragment used to determine intactness of the locus. Labeling of the figure is the same as for Figure 2. The YAC transgenes are displayed as solid lines. The ends of the lines coincide with the last hybridization signal observed and do not necessarily indicate YAC copy sequence endpoints. YAC copies that have 144 kb I-PpoI fragments should contain sequences extending to both the 5[prime] and 3[prime] I-PpoI sites of the [beta]-YAC because I-PpoI sites are very rare. Probes utilized for structural analyses are shown as dotted lines below the locus map and include a 3.1 kb BamHI 5[prime]HS5 fragment, a 0.8 kb EcoRI-PstI [beta]-globin 3[prime] enhancer fragment and a 3[prime]HS1 fragment (DF10; 47). If one or both I-PpoI sites are missing and because I-PpoI sites are extremely rare, we assume that these sequences are contiguous and that the locus is intact if all three probes hybridize to the same size fragment. Even if just 5[prime]HS5 and the 3[prime] [beta]-globin gene enhancer are detected, the locus should be considered to be functionally normal. Sequences found on fragments 50 kb or below are either on separate fragments or contain internal deletions, since the distance between 5[prime]HS5 and 3[prime]HS1 is 82 kb.

   A

   B

Figure 5. [beta]-YAC transgenic mice display position-independent, copy number-dependent [beta]-globin transgene expression. RNA was isolated from blood and subjected to RNAse protection analysis as described in Materials and Methods. Copy numbers were determined by the protocol described in Materials and Methods. Expression (percent human [beta]-globin mRNA/mouse [alpha]- + [zeta]-globin mRNA corrected for endogenous gene copy) is plotted on the y-axis versus transgene copy number on the x-axis. The diagonal line shows the result of linear regression analysis. (A) 248 kb [beta]-YAC. Each point is the mean of 2-8 F2, F3 or F4 littermates for each 248 kb [beta]-YAC line (lines 1, 3, 4, 7, 8, 10, 11). (B) Ppo-155 [beta]-YAC. Each point is the mean of 3-6 F2 littermates for each Ppo-155 [beta]-YAC line (lines 1-10), except for line 2, which had only one F2 progeny.

   A

   B

Figure 6. Expression of human [beta]-like globin transgenes in blood during development in 248 kb [beta]-YAC and 155 kb [beta]-YAC lines. RNA isolation, RNAse protection and quantitation were performed as described in Materials and Methods. The expression of each individual globin transgene is plotted as a percentage of total human globin gene expression ([epsis] + [gamma] + [beta]) on the y-axis. The development age of the fetus, in days, is displayed on the x-axis. Solid lines denote [epsis]-globin mRNA, dotted lines [gamma]-globin mRNA and dashed lines [beta]-globin mRNA. (A) 248 kb [beta]-YAC line 3 ([closed triangle]), 4 (×), 7 (+), 8 ( [closed square]), 10 ([closed triangle] ), 11 ([closed circle] ) and 12 ([open diamond]). (B) 155 kb [beta]-YAC line 1 ([closed diamond]) and 8 (×).

The Ppo-155 [beta]-YAC was used to generate [beta]-YAC transgenic mouse lines. Since the 144 kb I-PpoI fragment encompasses the entire cloned insert contained in the Ppo-155 [beta]-YAC, the detection of this fragment using any one single probe within the sequence spanned should be indicative of an intact YAC copy or copies. Screening for the presence of transgenes was accomplished in founder (F0) mice, using cells from the circulating blood and the approach described in Materials and Methods. Seven founders displayed an ~144 kb I-PpoI fragment indicating that the transgenes contained both I-PpoI sites. Of these, five had additional bands apparently resulting from integration of Ppo-155 [beta]-YACs that had lost one or both of their I-PpoI sites. Two of the remaining transgenics lacked hybridization signals, while five lines had bands of different size.

In two lines that contained only the 144 kb I-PpoI fragment (lines 1 and 8; Fig. 3B and 4), this fragment hybridized with all the probes from 5[prime]HS5 of the LCR through DF10, just downstream of 3[prime]HS1. These results indicate that if the 144 kb I-PpoI fragment is detected with any one probe, the YAC globin insert should be intact. This finding is expected because the 15 bp recognition sequence of this enzyme is extremely rare and the enzyme rarely cuts (e.g. I-PpoI cuts only once in an rDNA repeat sequence in chromosome 12 of the Saccharomyces cerevisiae yeast genome). Thus, it is unlikely that if one of the I-PpoI sites of the YAC is deleted, another I-PpoI site in the mouse genome will be juxtaposed precisely enough to generate a false-positive 144 kb fragment.

Detailed studies were done in 10 lines. Three probes spanning the [beta]-globin locus were used (Fig. 4) and signals from the 5[prime] end, middle and 3[prime] end of the locus were potentially detected. Two lines (lines 3 and 8) contained only the 144 kb I-PpoI fragment indicative of an intact YAC copy. Four lines (lines 1, 7, 9 and 10) contained this band and additional bands of altered size. The remaining four lines (lines 2, 4, 5 and 6) did not have the 144 kb I-PpoI fragment, but only fragments of altered size, indicating the presence of deletions. 5[prime]HS5 and 3HS1 sequences found on small fragments (lines 6 and 7) may represent internal deletions.

Correct structural analyses of YACs allows investigation of structure-function relationships of the transgenes

The detailed structural analyses described in the previous section allowed us to determine which globin genes were present in YACs having an intact LCR and therefore to measure [epsis]-, [gamma]- or [beta]-globin gene expression per copy for each gene.

The per copy expression of the adult [beta]-globin gene is shown in Figure 5. These analyses were done for [beta]-globin gene expression in adults. For each line we measured [beta]-globin mRNA levels per copy of transgene in several littermates (from 1 to 8 littermates; Tables 1 and 1). For correlation analysis the mean [beta]-globin mRNA level per line was calculated by averaging the levels of RNAse protection in several littermates. Correlation coefficients were r = 0.86, P < 0.01 for the 248 kb [beta]-YAC lines and r = 0.63, P < 0.05 for the 155 kb [beta]-YAC lines indicating a statistically significant correlation (Fig. 5). These results indicate that globin gene expression is copy number dependent as expected for normally functioning [beta]-globin loci.

Another way to examine whether expression of a transgene is position dependent or not involves calculation of the coefficient of variation (CV); CVs <0.5 indicate position-independent expression (14-16). For both the 248 and the 155 kb [beta]-YAC lines the CV was the same (0.26) indicating that globin gene expression is not influenced by the position of integration of the transgenes.

Additional evidence that the globin genes of YACs with an intact LCR function normally even when they sustain downstream or upstream deletions is derived from the developmental studies of the YAC transgenics. Human [beta]-like globin transgene expression was measured by RNAse protection during development in seven 248 kb [beta]-YAC lines carrying at least one intact [beta]-globin locus (lines 3, 4, 7, 8, 10, 11 and 12). In Figure 6A, the amounts of each individual human mRNA as a percentage of the total human mRNA are plotted versus the post-conception age of the fetus. Notice the consistency of the developmental globin gene switching pattern between the seven lines. The developmental profile of human globin transgene expression in two Ppo-155 [beta]-YAC lines is shown in Figure 6B. The expression pattern is essentially the same as that observed for the 248 kb [beta]-YAC lines. These data also indicate that the 3[prime] sequences present on the 248 kb [beta]-YAC (Fig. 2) and missing on the 155 kb [beta]-YAC (Fig. 4) and on 248 kb [beta]-YAC line 4 (Fig. 2) do not appear to play a role in globin gene switching of the transgenic mice.

Lines that do not contain the LCR do not express [beta]-like globin mRNAs at any stage of development (Fig. 2, line 6; 11). More recently, we have analyzed two additional 248 kb [beta]-YAC lines that lack the LCR (data not shown). One line contains a single copy of the YAC that spans the [epsis]-globin gene through the HPFH6 breakpoint. The other line contains two copies of the YAC, both of which extend from the [epsis]-globin gene through the HPFH3 breakpoint. Globin gene expression was not observed in either line at any stage of development by RNAse protection. Thus, proper gene expression and regulation requires the presence of the LCR in [beta]-YAC transgenics.

Variation in gene expression between littermates and between transgenic lines

The results of Tables 1 and 1 also show the small degree of intraline variation in globin gene expression in [beta]-YAC transgenes and that the variation between lines is not larger than the variation within lines.

Among the 248 kb [beta]-YAC transgenes, in three lines carrying one copy of the YAC, intraline variation in [beta]-globin gene expression ranged from 1.1- to 2.3-fold while interline variation was 1.8-fold. In the two copy lines, variation in expression within lines ranged from 1.0 to 1.7. The three lines differed up to 2.2-fold. In the four copy line variation in expression was 1.2-fold. Therefore, the maximal variation between lines carrying the same copy was more or less identical to that within lines.

Among littermates with one copy of the 155 kb [beta]-YAC, variation in [beta]-globin gene expression was 1.4-fold. In two copy lines, variation in expression ranged from 1.3 to 2.8. In three copylines, variation in expression ranged from 1.2 to 3.4. The maximal variation between lines with the same copy number was less than that within lines.

Table 1. Expression and copy number data for 248 [beta]-YAC transgenic lines
Line Copy number Expression/copya
among littermates		 Mean Standard deviation Coefficient of variation
1 2 160.0, 160.0 160.0 0 0
3 1 76.0, 72.0, 90.0,
139.0, 90.0,
61.0, 80.0		 86.9 25.1 0.29
4 1 136.0, 136.0, 120.0 130.7 7.5 0.06
7 1 134.0, 182.0,
147.0, 154.0,
170.0, 154.0		 156.8 16.9 0.11
8 4 99.8, 85.6, 99.8 95.1 8.2 0.08
10 2 61.0, 81.0, 61.0,
88.0, 67.0, 61.0,
101.5, 72.5		 74.1 14.9 0.20
11 2 129.5, 107.0,
107.0, 105.5,
84.5, 82.5, 85.0		 100.1 17.2 0.17
aPercent human [beta]/mouse [alpha] + [zeta]/copy number.

Table 2. Expression and copy number data for Ppo-155 [beta]-YAC transgenic lines
Line Copy number Expression/copya
among littermates		 Mean Standard deviation Coefficient of variation
1 3 129.0, 101.0,
74.0, 121.3		 106.3 24.6 0.23
2 3 140.7      
3 2 220.5, 163.5,
272.5, 262.0, 225.0		 228.7 42.9 0.19
4 2 145.5, 109.0,
88.0, 195.5, 246.0		 156.8 64.4 0.41
5 2 121.5, 194.0,
130.0, 124.5		 142.5 29.9 0.21
6 1 167.0, 209.0,
195.0, 185.0,
235.0, 178.0		 194.8 24.4 0.13
7 3 219.7, 85.7,
65.0, 81.7, 71.7		 104.8 64.8 0.62
8 2 89.0, 217.5, 81.5 129.3 76.4 0.59
9 3 145.3, 150.0, 128.7 141.3 11.1 0.08
10 2 101.5, 92.0, 116.5 103.3 12.4 0.12
aPercent human [beta]/mouse [alpha] + [zeta]/copy number.

DISCUSSION

Following the initial description of the YAC transgenics there have been several reports in which YAC transgenes have been used for analysis of gene function (7,17-31). In several of these studies only a few of the transgenic founders or lines expressed the expected clinical or molecular phenotypes, either because the absence of proper regulatory sequences led to expression of the integrated transgenes in a position dependent manner, or, most likely, because of structural rearrangements of the transferred YACs. In addition to YAC transgenics produced by pronuclear microinjection, two other methods have been used, lipofection of embryonic stem (ES) cells and yeast spheroplast-ES cell protoplast fusion (reviewed in refs 1,2). While these three methods have distinct advantages and disadvantages, the determination of structural integrity of integrated YACs is essential for all. Lipofection of ES cells or mouse erythroleukemia (MEL) cells with purified YAC DNA does not appear to enhance the recovery of transfectants containing intact YAC molecules (32). Conclusive structural data for YAC-bearing lines generated by spheroplast fusion is lacking, thus it is not known if this method results in conservation of YAC integrity in vivo. It should be noted however, that both of these methods require selection for G418 resistance to obtain YAC clones. This may predispose selection for YACs that have integrated into active chromatin, thus skewing functional data regardless of the structural condition of the integrated YACs.

A prerequisite of functional analysis of YAC transgenics is that all the necessary regulatory elements of a locus are included in the integrated transgene. A major problem with using YACs as transgenes is their tendency to fragment during in vitro handling or during the process of integration into the murine genome. Once the YAC DNA is no longer maintained in a solid matrix (i.e. agarose), it is subject to mechanical shear and degradation. YAC DNA is most vulnerable to these forces during dilution and filtration of the DNA solution just prior to microinjection and during the actual microinjection process itself. Therefore, it is tantamount for interpreting expression data that investigations involving YAC transgenes unambiguously demonstrate completeness and continuity of each integrated transgene copy in each established animal line. Otherwise, conclusions drawn from these data may be erroneous.

Our experience with human [beta]-globin locus YAC transgenics described in Results and data generated by other investigators (4,8,9,33) best illustrates the problem. Simply determining the presence of the YAC arms and testing for the presence of internal YAC sequences is not appropriate because positive hybridization signals do not necessarily mean that the integrated YAC is intact. The presence of recognition sites for a rare-cutting restriction enzyme in the YAC provides a convenient way to determine the structure of the integrated DNA as shown by the SfiI sites present within the 248 kb [beta]-globin locus YAC. Detailed structural studies are still essential because YACs with aberrant sizes still may contain all essential regulatory elements and genes of a locus. The situation becomes complex when no such sites for a rare-cutting enzyme are present. As shown in Results, this problem can be bypassed by retrofitting the YAC with rare or unique restriction enzyme sites. Also, retrofitting provides the ability to utilize the approach of detailed structural analysis we have outlined in Materials and Methods. I-PpoI (or other rare-cutting site) retrofitting of the YAC is an essential step when transgenic mice are used for definitive structure-function relationships of large loci. As we show in Results, another advantage in the use of the Ppo-retrofitted YACs is that structural studies are greatly simplified.

Other methods are useful for YAC structural determination in addition to that described here. Standard Southerns, PCR analysis, DNA sequence determination and repetitive DNA profiles are all ancillary methods that augment determinative structural analysis. Alternative methods that demonstrate individual YAC molecule continuity include RecA-assisted restriction enzyme cleavage and fiber fluorescent in situ hybridization. However, these protocols are technically difficult, expensive, time-consuming and not amenable to implementation for routine analysis of a number of transgenic lines.

It is likely that the degree of rearrangement sustained by YAC transgenes is related to the size of the YACs. All the lines we produced with the 248 kb [beta]-YAC contain rearranged or deleted YACs. In contrast, of the ten 155 kb [beta]-YAC lines, six contained at least one intact copy and two of these contained exclusively intact copies.

In spite of the presence of rearrangements, YACs that retained the major regulatory element of the [beta]-globin locus, the LCR, displayed quantitatively normal expression of the genes of the locus. Also, the developmental profile of globin gene expression was normal. Normal function of the LCR is characterized by copy number-dependent and site of integration-independent globin gene expression. Both the 248 and 155 kb [beta]-YAC lines are characterized by position-independent and copy number-dependent expression indicating that in both instances, function of the LCR is normal. These results demonstrate that the YACs function normally, independently of where they are integrated in the murine genome. However, [beta]-like globin gene expression was not detected at any stage of development in three 248 kb [beta]-YAC lines lacking the LCR, but containing the remainder of the locus (Fig. 2, line 6 and data not shown). In contrast, transgenic mice generated with a 40 kb G[gamma]-A[gamma]-[psi][beta]-[delta]-[beta] gene cluster or a 13 kb G[gamma]-A[gamma] fragment, neither of which contained the LCR, showed developmentally regulated expression of the human [gamma]- and [beta]-globin transgenes (34,35). It is possible that sequences present in the YAC, but absent in these smaller fragments modulate globin gene expression resulting in locus silencing when the LCR is missing. Thus, when all the genes and flanking sequences of the [beta] locus are intact, the LCR is essential for globin gene expression throughout development.

The results obtained with the [beta]-globin locus YACs should be contrasted with those obtained with a 70 kb [beta]-globin locus linked cosmid construct in which position-dependent gene expression has been reported (36,37). It is likely that this influence of the position of integration on the function of the 70 kb globin transgene reflects the absence of the upstream and downstream sequences that flank the locus and are present in both the 248 and 155 kb [beta]-YACs. Perhaps YACs allow better function of regulatory elements, like the LCR, through sequences that bracket a locus and decrease the impact of deleterious effects from the heterochromatin.

MATERIALS AND METHODS

PpoI retrofitting of the 155 kb [beta]-YAC

The 150 kb [beta]-YAC A201F4 (9) was retrofitted to contain I-PpoI sites at the insert-vector junction and a PGKneo selectable gene cassette by the method of Fairhead et al. (13). The resultant YAC is 5 kb larger and is referred to as the Ppo-155 [beta]-YAC.

YAC purification and microinjection

The 248 kb [beta]-YAC was purified as previously described (5,11,38,39). The 155 kb [beta]-YAC was purified with modifications to the original protocol. Briefly, preparative agarose blocks containing high-mass DNA were prepared from yeast carrying the 155 kb [beta]-YAC (38). DNA was fractionated by PFGE using a CHEF DRII apparatus (Bio-Rad, Hercules, CA) under the following conditions: 0.5% agarose MP gel (Boehringer Mannheim, Indianapolis, IN), 0.5× TBE (45 mM Tris, 45 mM boric acid, 1 mM EDTA), 200 V, 60 s switch, 18-24 h, at 12°C. Marker lanes and the edges of the preparative lane were cut and stained with ethidium bromide to locate the position of the YAC. Notches were made in these stained gel pieces to denote the location of the 155 kb [beta]-YAC and the gel was reassembled. Using the notches as guides, the region of the preparative lane containing the [beta]-YAC was cut from the gel, as was a region containing one of the yeast chromosomes. The [beta]-YAC gel slice and the yeast chromosome slice were rotated 90° relative to their original direction of electrophoretic migration and subjected to electrophoresis in 4% low-melting point agarose (LMPA; NuSieve GTG, FMC, Rockland, ME), 0.5× TBE at 47 V for 16 h to concentrate the YAC DNA. The yeast chromosome lane was stained with ethidium bromide to determine the migration distance of the [beta]-YAC DNA into the LMPA gel. The region containing the YAC DNA was excised from the gel, weighed and equilibrated in 40-100× vol of injection buffer (10 mM Tris pH 7.5, 250 µM EDTA pH. 8.0, 100 mM NaCl) for 1 h at room temperature. The gel slice was then placed in a microfuge tube, the agarose was melted at 68°C for 10 min and the tube was rapidly transferred to a 42.5°C water bath and equilibrated at this temperature for 5 min. Two units of [beta]-agarase I (New England Biolabs, Beverly, MA) were added per 100 mg melted gel and the agarose was digested overnight at 42.5°C. The sample was placed on ice for 10 min to determine if digestion was complete. Concentration of the YAC DNA was determined by standard agarose gel electrophoresis using [lambda] HindIII markers as concentration standards and integrity was determined by PFGE (5,6). [beta]-YAC DNA was diluted to 2 ng/µl in injection buffer and filtered through a 0.22 µm Acrodisk (Gelman Sciences, Ann Arbor, MI) prior to microinjection of fertilized oocytes.

Use of blood buffy coat-derived plugs for PFGE analysis

One to two hematocrit tubes of blood were drawn from the tail vein of each founder animal and the buffy coat (the nucleated cell fraction of whole blood) was collected following centrifugation of the whole blood. The concentrated cell pellet was resuspended in 10 µl agarose and the micro-plug was used to prepare agarose blocks containing high-mass DNA as described in the preceding section. The resultant plug is sufficient for one or two restriction enzyme digests. Restriction enzyme digestion, PFGE and Southern blot analysis were performed as described below. A spot of hybridization may be observed in each lane. When compared with an SfiI- or I-PpoI-digested human [beta]-globin locus YAC standard it is possible to identify which founders contain an intact [beta]-globin locus or loci. Founder mice judged to have intact [beta]-globin loci were used to establish lines.

Structural analysis of integrity of [beta]-YAC transgenics

PFGE conditions and Southern blot hybridization conditions were as previously described (2,5,6). An outline of our approach to structural analysis is as follows. A single cell suspension was made from the liver of a transgenic mouse. The suspension was used to prepare agarose blocks containing high-mass genomic DNA. Portions of the blocks were digested with SfiI or I-PpoI. Up to 13 lanes of an agarose gel were loaded with digested agarose block slices and the DNA was fractionated by PFGE. The DNA was transferred to a membrane by Southern blot. The individual lanes were cut from the blot and hybridized to separate probes that span the [beta]-globin locus domain. After washing, the blot was reassembled and an autoradiograph made. Individual YAC copies were visualized and the extent of the locus present for each copy was deduced. When probes do not show signals, that region is not present on the YAC. Copies with sizes larger or smaller than 140 kb for SfiI digests or 144 kb for I-Ppo-I digests may contain the complete [beta]-globin locus, but lack one or both flanking sites. Thus, the size of the fragment is determined by the juxtaposition of murine genomic SfiI or I-PpoI sites near transgene sequences.

Copy number determination

Although the pulsed-field gels show the different size YAC molecules that reside within a transgenic line, they do not necessarily give an accurate determination of copy number because there may be more than one copy of a YAC of an observed size. Thus, to conclusively determine copy numbers and provide accurate structural data, it is important to include a standard copy number analysis to corroborate the PFGE data. Copy numbers were determined as described (2,12).

RNAse protection analysis

RNA isolation, probe template DNA preparation, probe-labeling and RNAse protection were performed as described (5,6,11,40). Total RNA was isolated from the yolk sac, liver and blood of transgenic F2 staged fetuses using the method of Chomczynski and Sacchi (41). Antisense RNA probes for human [epsis], [gamma] and [beta] and murine [alpha] and [zeta] mRNA were synthesized by in vitro transcription from linear templates using T7 RNA polymerase (42). Template DNAs were pT7[epsis] (188), pT7A[gamma]m (170), pT7[beta]m, pT7M[alpha] and pT7M[zeta] (43-46). RNA was hybridized overnight at 47°C with 106 c.p.m. of each radiolabeled probe. RNAse protections were quantitated by phosphorimaging.

ACKNOWLEDGEMENTS

We thank Mary Eng, Harauld Haugen, Betty Josephson, Alex Rohde and Sara Shaw for excellent technical assistance. This work was supported by National Institutes of Health grants DK45365 and HL53750.

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*To whom correspondence should be addressed at present address: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA. Tel: +1 913 588 6907; Fax: +1 913 588 7440; Email: kpeterson@kumc.edu

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